Bioluminescent Musculoskeletal Wound Model

This is an enhanced PDF from The Journal of Bone and Joint Surgery The PDF of the article you requested follows this cover page.

Comparison of Bulb Syringe and Pulsed Lavage Irrigation with Use of a Bioluminescent Musculoskeletal Wound Model Major Steven J. Svoboda, Terry G. Bice, Heather A. Gooden, Daniel E. Brooks, Darryl B. Thomas and Joseph C. Wenke J. Bone Joint Surg. Am. 88:2167-2174, 2006. doi:10.2106/JBJS.E.00248

This information is current as of April 2, 2007 Supplementary material

Commentary and Perspective, data tables, additional images, video clips and/or translated abstracts are available for this article. This information can be accessed at http://www.ejbjs.org/cgi/content/full/88/10/2167/DC1

Reprints and Permissions

Click here to order reprints or request permission to use material from this article, or locate the article citation on jbjs.org and click on the [Reprints and Permissions] link.

Publisher Information

The Journal of Bone and Joint Surgery 20 Pickering Street, Needham, MA 02492-3157 www.jbjs.org

Downloaded from www.ejbjs.org on April 2, 2007

Svoboda.fm Page 2167 Wednesday, September 6, 2006 2:09 PM

2167 COPYRIGHT © 2006

BY

THE JOURNAL

OF

BONE

AND JOINT

SURGERY, INCORPORATED

Comparison of Bulb Syringe and Pulsed Lavage Irrigation with Use of a Bioluminescent Musculoskeletal Wound Model BY MAJOR STEVEN J. SVOBODA, MD, TERRY G. BICE, MS, HEATHER A. GOODEN, BS, DANIEL E. BROOKS, BS, DARRYL B. THOMAS, MD, AND JOSEPH C. WENKE, PHD Investigation performed at the United States Army Institute of Surgical Research, Fort Sam Houston, Texas

Background: Despite the fact that wound irrigation is a common surgical procedure, there are many variables, including delivery device, irrigant type, and fluid volume, that have yet to be optimized. The purpose of this study was to compare, with use of transgenic bioluminescent bacteria and standard quantitative microbiological methods, the efficacy of pulsed lavage and bulb syringe irrigation in reducing wound bacterial counts. Methods: A caprine model of a complex, contaminated musculoskeletal wound was developed with use of a bioluminescent strain of Pseudomonas aeruginosa that can be quantified. Luminescent activity was recorded as relative luminescent units with use of a photon-counting camera six hours after the wound was created and inoculated. Twelve goats were randomly assigned to either the pulsed lavage group or the bulb syringe irrigation group. Each wound was irrigated with normal saline solution in 3-L increments for a total of 9 L and was imaged after each 3-L increment. In addition, quantitative culture samples were obtained from different tissues within the wound before and after irrigation. Results: Pulsed lavage decreased the amount of relative luminescent units by 52%, 64%, and 70% at 3, 6, and 9 L, respectively. The bulb syringe irrigation reduced the amount of relative luminescent units by 33%, 44%, and 51% at these same time-points. Significant differences in luminescence were noted between the two groups after both 6 and 9 L of irrigation (p ≤ 0.04). The correlation coefficients between relative luminescent units and quantitative cultures for the condition before irrigation and after irrigation were r = 0.96 and 0.83, respectively. Conclusions: Pulsed lavage was more effective than bulb syringe irrigation in reducing bacterial luminescence after both 6 and 9 L of irrigation. Both device and volume effects can be demonstrated with use of this model. Bioluminescent bacteria provide a method to visualize bacterial distribution and to quantify the bacteria in a wound. Clinical Relevance: Pulsed lavage is a more effective and efficient method of irrigation to remove bacteria in a complex musculoskeletal wound. In the model we used, pulsed lavage irrigation with 3 L of saline solution resulted in a reduction of approximately the same amount of bacteria as did irrigation with 9 L with use of a bulb syringe.

I

rrigation is an integral step in the management of softtissue injuries and open fractures, typically following débridement of the injured soft tissues. Irrigation is often a dogmatic step in initial wound management, yet several factors including fluid type, fluid volume, and delivery method must be considered prior to wound irrigation. Recommended fluid volumes have ranged from 6 to 10 L1-3. With regard to fluid types, normal saline solution is the standard to which all A commentary is available with the electronic versions of this article, on our web site (www.jbjs.org) and on our quarterly CD-ROM (call our subscription department, at 781-449-9780, to order the CD-ROM).

other irrigants have been compared1,4-8. Additives such as antibiotics7-14, surfactants6, soaps15, and antiseptics1,7 have all been investigated with mixed results. Device comparisons have been performed, with bulb syringe irrigation being the usual standard to which gravity flow16,17, pulsed lavage18, and other high-pressure lavage devices are compared1,19. While pulsed lavage is perhaps the most common device used in irrigation, it has been implicated as causing foreign bodies and bacteria to be driven further into the tissues3,20,21, and it may result in injury to chondrocytes and osteoblasts22-25. The superiority of high-pressure devices (e.g., pulsed lavage) over low-pressure devices (e.g., bulb syringe) is



2168 THE JOURNAL OF BONE & JOINT SURGER Y · JBJS.ORG VO L U M E 88-A · N U M B E R 10 · O C T O B E R 2006

not conclusive and may depend on different factors such as the degree of initial wound contamination26. The definition of what exactly constitutes high and low pressure is not well established in the literature. However, it is generally accepted that low pressure is 35 psi (241.3 kPa)21. The so-called gold-standard method of measuring efficacy has been quantitative culture, which is a consumptive, tissue-destructive process. Loss of tissue can be minimized by taking fewer and smaller biopsy samples, but this limits the culture yield. In addition, quantitative culture samples only a small portion of the wound and may omit a large area of localized contamination or infection. The ability to determine the distribution and quantity of bacteria in a wound is desirable, especially for the purpose of improving techniques and equipment for irrigation. Transgenic bacteria have been developed with the gene for luminescence spliced into their own genome. These bioluminescent bacteria produce light that can be detected with a high-sensitivity photon-counting camera. Therefore, the relative quantity and distribution of the bacteria in the wound can be determined. The purpose of this study was to determine whether pulsed lavage irrigation is more effective than bulb syringe irrigation in removing bacteria from a contaminated wound in a large-animal model. We hypothesized that pulsed lavage, because of the higher pressures it generates, would remove more of the bacteria from the wound than would bulb syringe irrigation. Materials and Methods General ll procedures were performed in a laboratory accredited by the Association for Assessment and Accreditation of Laboratory Animal Care after approval of the protocol was obtained from the Institutional Animal Care and Use Committee.

A

Bacterial Preparation Pseudomonas aeruginosa (ATCC 27317) was genetically engineered to be luminescent by random chromosomal insertion of the luciferase-luciferin construct luxCDABE obtained from Photorhabdus luminescens, a nematode symbiont bacterium27. With the lux gene incorporated into the organism, it was then referred to as Pseudomonas aeruginosa (lux). This bioluminescence became a stable, heritable genetic trait. For each inoculation procedure, the stock culture was grown up over eighteen to twenty hours and was diluted to 108 colony-forming units (CFU)/mL in 0.85% NaCl. The concentration was confirmed by performing a plate count with use of the spread plate technique. Surgical Procedure—Wound Creation Twelve castrated, adult male Spanish-Boer goats (Capra hircus) (Talley Ranch, Uvalde, Texas) were fasted for twenty-four hours, and water was withheld for twelve hours prior to surgery. Anesthesia was induced with a combination of ketamine hydrochloride (2.2 to 7.0 mg/kg) and midazolam (0.125 to 0.250 mg/kg) administered intravenously through a 21-gauge

C O M P A R I S O N O F B U L B S Y R I N G E A N D P U L S E D L AV A G E I R R I G A T I O N I N A M U S C U L O S KE L E T A L WO U N D M O D E L

needle. After endotracheal intubation was done, anesthesia was maintained with isoflurane and supplemental oxygen. An epidural injection of morphine (0.1 mg/kg) diluted in 0.9% sterile saline solution to a volume of 0.13 mL/kg was given both as an adjunct to general anesthesia and for its durable postoperative analgesic effect. The goat was placed supine on the operating table, and the left lower extremity was shaved and aseptically prepared. The limb was then draped free with a sterile stockinette over the hoof and lower leg. The tibial tubercle was marked, and a 5-cm skin incision approximately 1 cm lateral to, and at the level of, the tubercle was made and extended distally to the level of the medial periosteum and fascia overlying the anterior and lateral leg compartments. The lateral compartment was elevated from its attachment to the lateral aspect of the tibia after the lateral compartment fascia was incised with use of electrocautery. The fascia was also elevated from the superficial surface of the anterior and lateral compartments. The medial tibial periosteum was exposed and incised longitudinally throughout the length of the skin incision and parallel to the incision in the anterior compartment fascia. This incision was measured so that a 6-mm strip of periosteum was left intact on the anteromedial aspect of the tibia. The more posterior portion of periosteum was elevated with a blunt periosteal elevator and retracted medially. A partial medial cortical injury measuring 1.2 cm in diameter was created in the tibia with use of a 3-mm drill-bit on a twist drill and a small osteotome. Care was taken to avoid breaching the cortical wall and entering the medullary canal. Three Kelly clamps were spaced evenly over a 5-cm segment of the anterior compartment muscles and were left in place for three minutes to induce a standardized crush injury to the anterior compartment muscles. Concurrently, electrocautery was used to create thermal damage to the intervening muscle between the clamps. Thus, the wound rendered was complex, involving injury to muscle, fascia, periosteum, and bone (Fig. 1). The wound was inoculated with 1 mL of >108 CFU/mL of Pseudomonas aeruginosa (lux), which was spread evenly over the wound surfaces with a cotton-tipped applicator soaked in the same inoculum. The wound was left open for a five-minute period after which it was dressed open with a cover sponge, a rolled gauze dressing, and Vetrap bandaging tape (3M Animal Care Products, St. Paul, Minnesota). Postoperative Care After surgery, the goats recovered in their pens and were allowed activity ad libitum. If an animal demonstrated any discomfort, a fentanyl citrate patch (Duragesic 50) was placed on the neck area to control postoperative pain. The goats were killed six hours after inoculation with a concentrated solution of pentobarbital sodium (90 mg/kg administered intravenously). Six hours was chosen as the incubation time because luminescence at six hours was visible and measurable by the camera and the removal of bacteria by means of irrigation was still possible. In addition, the current standard of care dictates that the initial débridement and irrigation should be per-




formed within a few hours after the injury3. Moreover, in a recent clinical trial, the average interval between the time that an open fracture was sustained to the time of irrigation was approximately six hours8. Imaging Procedure Bioluminescent bacteria emit light in proportion to their number28,29. This allowed the use of a specialized imaging system contained in a light-free enclosure to quantify the amount of bacteria in the wound. A photon-counting camera (Charge Couple Device [CCD] Imaging System Model C2400; Hamamatsu Photonics, Hamamatsu-City, Japan), which recognizes the event created by a single photon hitting the photocathode of an image intensifier, was used. By accumulating many images containing binary photon information, a luminescent image is generated. Superimposition of this image onto a gray-scale background image yields information on the location and intensity in terms of photon number. The camera was connected to a computer system with Windows 98 (Microsoft, Redmond, Washington) through an image processor (model C5510, Argus-20; Hamamatsu Photonics). Argus-20 Interface software (version 1.10; Hamamatsu Photonics) and AquaCosmos basic software (version 1.30; Hamamatsu Photonics) were used to acquire images and to process the image data collected. Each day, prior to imag-

Fig. 1

A complex wound involving injury to muscle, fascia, periosteum, and bone was created.


TABLE I Percentage Decrease in Relative Luminescent Units After Irrigation* Volume of Saline Solution Used in Irrigation 3L

6L

9L

Bulb syringe

Treatment Group

33 ± 7

44 ± 7

51 ± 7

Pulsed lavage

52 ± 9

64 ± 8

70 ± 8

0.06

0.03

0.04

Significance (p value)

*The values are given as the mean and the standard error of the mean.

ing, a background noise image was made to ensure that background light was kept below a known minimum level. The goat was placed supine on the operating table within the light-free enclosure. The camera was placed directly over the wound, and the leg was secured to the camera apparatus through an adapted external fixation system (Synthes, Paoli, Pennsylvania) with use of two 5-mm external fixator pins placed as far proximally and distally as possible from the wound location. The hip and knee joints were flexed to 90° in the imaging apparatus so that the wound surface was 9 cm directly beneath the camera lens. Gelpi retractors were placed at the proximal and distal wound margins. A black-and-white image of the wound was made, and this was followed by a photon count of the same region. This entire wound photon count was quantified as relative luminescent units and was displayed in a pseudospectrum ranging from red (most intense) to blue (least intense), with black representing no photon detection. At this time, locations in the periosteum, fascia, and anterior muscle compartment with the most photon detection were identified. Each of the three regions was marked by a surgical staple (Visistat; Weck Closure Systems, Research Triangle Park, North Carolina), and an additional sequence of black-and-white and photon images was taken to confirm staple placement. A 6-mm-square softtissue biopsy sample was taken from above each staple with use of a number-11 scalpel blade (Bard-Parker; Becton Dickinson, Franklin Lakes, New Jersey). In the case of the fascia and periosteum, full-thickness samples of each tissue were harvested. Approximately 4-mm-thick sections of the muscle were harvested for quantitative cultures. Irrigation Sequence The goats were randomly assigned to either the group that had irrigation with the bulb syringe (Kendall, Mansfield, Massachusetts) or the group that had pulsed lavage (InterPulse irrigation system; Stryker Instruments, Kalamazoo, Michigan) such that both groups consisted of six goats. Each wound was then sequentially irrigated with 3 L of normal saline solution with the selected device for a total of 9 L. Device tips were held 1 cm from the wound surface, and the pulsed lavage device was operated at its highest setting. The pulsed lavage system used a high-flow tip attachment (model 210-14) with a maxi-



2170 C O M P A R I S O N O F B U L B S Y R I N G E A N D P U L S E D L AV A G E I R R I G A T I O N I N A M U S C U L O S KE L E T A L WO U N D M O D E L

THE JOURNAL OF BONE & JOINT SURGER Y · JBJS.ORG VO L U M E 88-A · N U M B E R 10 · O C T O B E R 2006

TABLE II Quantative Culture Results by Tissue Location* Before Irrigation

After Irrigation

Treatment Group

Periosteum

Muscle

Fascia

Periosteum

Muscle

Fascia

Bulb syringe

2.64 ± 1.1

1.28 ± 1.12

5.10 ± 2.29

1.34 ± 0.56

1.11 ± 0.83

1.77 ± 1.33

Pulsed lavage

3.25 ± 1.7

1.27 ± 0.77

1.52 ± 0.99

1.38 ± 0.85

0.02 ± 0.01

0.55 ± 0.34

8

*The values are given as the mean colony-forming units (CFU/g [10 ]) and the standard error of the mean.

mum pressure of 19 psi (131 kPa) (minimum, 6 psi [41.4 kPa]) and maximum flow rate of 1025 mL/min. After each 3-L increment, both a black-and-white and a photon-count image were made. After images were made of the condition following irrigation with 9 L of saline solution, tissue samples were harvested from each of the three locations inferior to the previously placed surgical staple for quantitative cultures. Data Analysis Raw data were collected in the form of relative luminescent units generated by the charge-coupled device camera and image processor as well as from quantitative cultures. All data were saved in Excel XP software (Microsoft). For the relative luminescent units, the AquaCosmos imaging software provided a count of relative luminescent units for the entire field within view of the camera. The quantitative culture results were expressed in terms of CFU/mL and were converted to CFU/g of tissue. Scatter plots were created of relative luminescent units

compared with the average CFU/g of the periosteum, fascia, and muscle. Linear regression was performed, and correlation coefficients were calculated. Ratios of relative luminescent units after each volume of irrigation compared with the baseline relative luminescent units were calculated to include standard errors of the mean. A pre hoc power analysis demonstrated that six goats per group were required to achieve beta of 0.80 and an ability to identify a 25% difference between the bulb syringe and pulsed lavage groups with respect to the ratio of the relative luminescent units after irrigation to the baseline relative luminescent units. Alpha was set at 0.05, and data are presented as the mean and the standard error of the mean. All ratios between the bulb syringe and pulsed lavage groups were analyzed with use of a hierarchical mixed-model analysis of variance. Post hoc testing was performed with t tests for further within-group comparisons of ratios with use of the Bonferroni correction. SAS statistical software (SAS Institute, Cary, North Carolina) was used for all statistical calculations.

Fig. 2

Comparison of the mean ratio of luminescent bacteria remaining in the wound following 3-L iterations of bulb syringe irrigation compared with pulsed lavage irrigation. The solid black column (Pre) represents the baseline luminescence detected prior to irrigation. P values indicate significant differences in volumes within groups. An asterisk indicates a significant difference between pulsed lavage irrigation and bulb syringe irrigation at 6 and 9 L (p = 0.03 and p = 0.04, respectively).





Fig. 3

A: Image of the luminescent bacteria within a wound before irrigation, that is, baseline luminescence. B: Image of the luminescent bacteria remaining in the wound after 9 L of pulsed irrigation.

Results ulsed lavage decreased bacterial luminescence more than bulb syringe irrigation with use of 6 and 9 L of saline solution (p ≤ 0.04), but no difference was found after 3 L of irrigation (p = 0.06) (Table I). With 9 L of irrigation, bulb syringe irrigation reduced the bioluminescence by 51%, whereas a 70% reduction was seen with use of pulsed lavage. With quantitative cultures, similar decrements were observed after 9 L of irrigation (Tables II and III). A within-group comparison of the percentage of relative luminescent units reduced following the various volumes of irrigation showed significant decreases

P

(p ≤ 0.002) in the ratio of the remaining luminescence between the two groups at all irrigation volumes tested (Fig. 2). The correlation coefficients for the relative luminescent units compared with the average CFU/g of the periosteum, muscle, and fascia were 0.96, 0.83, and 0.92 for the condition before irrigation, the condition after irrigation, and the pooled data, respectively. Qualitative analysis indicated that the bacterial luminescence was not homogeneous throughout the wound (Fig. 3). However, there was not a distinguishable pattern regarding which area or tissue within the wound had the highest amount of bacterial luminescence.




TABLE III Overall Quantitative Culture Results* Treatment Group

Before Irrigation

After Irrigation

Bulb syringe

9.02 ± 1.81

4.22 ± 0.85

Pulsed lavage

6.03 ± 1.34

1.95 ± 0.45

*The values are given as the mean colony-forming units (CFU/g [108]) and the standard error of the mean.

Discussion novel model of a contaminated orthopaedic wound was created to test the hypothesis that irrigation with pulsed lavage would be more effective than bulb syringe irrigation in removing bacteria from a contaminated wound. The use of bioluminescent bacteria allowed the changes in quantity and distribution of bacteria in the entire wound to be visualized and quantified noninvasively over multiple volumes of irrigant. The use of bioluminescent bacteria as a research tool has a relatively brief but successful history. Bacterial bioluminescence and bacterial quantity had correlation coefficients of 0.98 and 0.99 in a mouse foreign-body model and a mouse hind-limb infection model28,29. The previous studies with use of bioluminescent bacteria to create contaminated wounds either consumed the entire sample of tissue after imaging28 or utilized an implanted foreign body 29. The overall relative luminescent units compared with bacterial count correlation coefficient from the current study (0.92) compares extremely well with the correlation coefficients reported for the other biologic models, especially considering that the current musculoskeletal wound imaged was more complex than that evaluated in the mouse models. Many studies have compared bulb irrigation and pulsed lavage irrigation; however, none have provided conclusive proof of the superiority of pulsed lavage over bulb syringe irrigation in a contaminated musculoskeletal wound with use of a pulsed lavage device that produces pressures similar to those routinely used in the treatment of extremity injuries. In an in vitro model of stainless-steel screws coated with slime-producing bacteria, Anglen et al.4 subjected the screws to either bulb irrigation or pulsed lavage irrigation. After irrigation with 1 L of normal saline solution, the decrease in bacterial number was 100 times greater with pulsed lavage than with bulb syringe irrigation. This is a compelling difference; however, it did not assess bacterial clearance in an actual wound. In another study, a small-animal model of a paraspinal contaminated wound demonstrated that pulsed lavage removed more bacteria than did bulb syringe irrigation16. The pulsed lavage used in that study, a 50 psi (345 kPa) Water Pik device (Water Pik Technologies, Newport Beach, California), produced more pressure than that available in the commercial device that we tested and that is commonly used to irrigate wounds. In a study with a similar rat model, Hamer et al.30 compared gravity flow, bulb syringe, and pulsed lavage in wounds contaminated with both Staphylococcus aureus

A


and Escherichia coli. The authors concluded that pulsed lavage reduced bacterial culture counts and clinical infection rates more than gravity flow and bulb syringe irrigation. It should be noted that the pulsed lavage device used in their study also produced 50 psi (345 kPa). Unfortunately, the results from these studies cannot be directly extrapolated to clinical wound care either because the models used were not relevant to trauma-related musculoskeletal injuries that are routinely débrided and irrigated or because the pulsed lavage devices used in the studies generated pressures well above what is routinely used in the operating room during irrigation (approximately ≤20 psi [≤137.9 kPa] with pulsed lavage irrigation and approximately 1 psi [6.9 kPa] for bulb syringe irrigation). Both a volume effect and a device effect are evident when bacterial luminescence counts are compared before irrigation and after irrigation. The volume effect is demonstrated by the observation that there is a significant difference in relative luminescent units between each volume within each device group (p ≤ 0.02). However, it appears that the amount of reduction in bacterial luminescence diminishes with each 3-L iteration (Fig. 2). This volume effect has been seen clinically as well. Gustilo and Anderson31 observed that rates of infection in open fractures decreased with increasing volumes of irrigation. Conversely, in an in vitro model, Gainor et al.32 noted no significant decrease in bacterial counts after irrigation with 100 mL to 1 L of normal saline solution or after irrigation with 1 to 10 L. In that study, bacteria were placed on strips of beef and were allowed fifteen minutes of incubation before irrigation with a Water Pik (Water Pik Technologies). The lack of volume effect seen in that study may be attributed to two factors: (1) the short incubation time, which did not allow for bacterial adhesion, and (2) the high-pressure pulsatile device. The six-hour incubation period that was chosen for our study more closely mimics the current standard of care for this type of injury. In some scenarios, the appropriate irrigation volume to be used may depend not only on the severity and contamination of the wound (which would dictate that high volumes be used) but also on the resources available. The possible need to ration available irrigant solutions while caring for combat casualties18 or domestic mass casualties only underscores the practical need to understand the effectiveness of different irrigation techniques over various volumes. In this light, the device effect becomes more significant as demonstrated by the observation that the pulsed lavage reduced bacterial luminescence significantly more than the bulb syringe at volumes of 6 L and 9 L (p ≤ 0.04). After 3 L of irrigation, the pulsed lavage decreased luminescence by 52% and bulb syringe irrigation decreased it by 33% of baseline. With only six animals in each group, these amounts were not significantly different (p = 0.06). The first 3 L of pulsed lavage irrigation and 9 L of bulb syringe irrigation reduced luminescence by 52% and 51%, respectively (Table I). The similar reduction in bacteria implies that pulsed lavage irrigation at lower volumes may be as effective as higher volumes with use of the bulb syringe. Consider-




ing that resources are limited in austere environments, such as forward surgical teams on the battlefield, these data suggest that pulsed lavage devices may be more desirable despite the fact that they weigh more and are more expensive than bulb syringes. Qualitatively, this model provides data regarding the spatial distribution of bacteria not previously possible with quantitative cultures (Fig. 3). The distribution of luminescence was not homogeneous, although the bacteria were evenly applied initially over the entire surface area of the wound. A pattern could not be discerned to describe which type of tissue allowed the most bacterial adherence or best growth. This asymmetric distribution supports the notion that typical quantitative sampling methods have an inherent degree of error, rendering their culture results less reliable. This finding should lend caution to the conclusions provided by studies that use quantitative cultures solely to assess bacterial load. The current study differs from the standard of care for contaminated wounds because it did not include débridement, and this may be considered a weakness. We support débridement as an essential step in open wound management; however, its inclusion in this study would have added additional procedural variability. Although the predominant bacterial species present during initial wound management are generally gram-positive, gram-negative bacteria, such as Pseudomonas aeruginosa, are also present up to 33% of the time at the initial débridement of open fractures31. During model development, we attempted to use grampositive strains, but the bacteria did not emit enough photons to capture using our current camera system. Care should be taken in extrapolating the current results to conditions involving other organisms until other bacterial species have been used in the model. Another limitation of our study is that it was acute in nature and did not follow contaminated wounds for any time after the initial irrigation to evaluate for signs of clinical infection. It is possible that the pulsed lavage, due to the higher pressure (19 psi [131 kPa]), may have propelled the bacteria into the tissue, perhaps resulting in an increased rate of clinical infection. The same


pulsed lavage device has been shown to drive bacteria into soft tissue in an ex vivo model21. Pulsed lavage could have also increased local inflammation or destroyed viable tissue; neither was assessed in the present study. Ongoing studies at our institution with use of bioluminescent gram-positive organisms, as well as survival studies that allow the wounds to be followed for clinical signs of infection, are planned. Further study is also ongoing with regard to the evaluation of irrigant types and additives as well as device prototypes. Transgenic bioluminescent bacteria provide a powerful tool for the noninvasive study of extremity wound injuries. Irrigation with pulsed lavage is more effective than irrigation with a bulb syringe in this large-animal model of a complex, contaminated musculoskeletal wound and may allow smaller volumes of irrigant to be used. Major Steven J. Svoboda, MD Terry G. Bice, MS Heather A. Gooden, BS Daniel E. Brooks, BS Darryl B. Thomas, MD Joseph C. Wenke, PhD United States Army Institute of Surgical Research, 3400 Rawley E. Chambers Avenue, Fort Sam Houston, TX 78234-6315. E-mail address for J.C. Wenke: [email protected] The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of Defense or United States government. The authors are employees of the United States government. This work was prepared as part of their official duties and, as such, there is no copyright to be transferred. The authors did not receive grants or outside funding in support of their research for or preparation of this manuscript. They did not receive payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated. doi:10.2106/JBJS.E.00248

References 1. Anglen JO. Wound irrigation in musculoskeletal injury. J Am Acad Orthop Surg. 2001;9:219-26. 2. Swiontkowski MF. The multiply injured patient with musculoskeletal injuries. In: Rockwood CA, Green DP, Bucholz RW, Heckman JD, editors. Rockwood and Green’s fractures in adults. 4th ed. Philadelphia: Lippincott-Raven; 1996. p 121-58. 3. Olson SA, Willis MD. Initial management of open fractures. In: Bucholz RW, Green DP, Heckman JD, Rockwood CA, Court-Brown CM, editors. Rockwood and Green’s fractures in adults. 6th ed. Philadelphia: Lippincott Williams and Wilkins; 2006. p 389-424. 4. Anglen JO, Apostoles S, Christensen G, Gainor B. The efficacy of various irrigation solutions in removing slime-producing Staphylococcus. J Orthop Trauma. 1994;8:390-6. 5. Conroy BP, Anglen JO, Simpson WA, Christensen G, Phaup G, Yeager R, Gainor BJ. Comparison of castile soap, benzalkonium chloride, and bacitracin as irrigation solutions for complex contaminated orthopaedic wounds. J Orthop Trauma. 1999;13:332-7.

6. Marberry KM, Kazmier P, Simpson WA, Christensen GD, Phaup JG, Hendricks KJ, Anglen JO, Gainor BJ. Surfactant wound irrigation for the treatment of staphylococcal clinical isolates. Clin Orthop Relat Res. 2002;403:73-9. 7. Howell JM, Stair TO, Howell AW, Mundt DJ, Falcone A, Peters SR. The effect of scrubbing and irrigation with normal saline, povidone iodine, and cefazolin on wound bacterial counts in a guinea pig model. Am J Emerg Med. 1993;11:134-8. Erratum in: Am J Emerg Med. 1993;11:319. 8. Anglen JO. Comparison of soap and antibiotic solutions for irrigation of lowerlimb open fracture wounds. A prospective, randomized study. J Bone Joint Surg Am. 2005;87:1415-22. 9. Benjamin JB, Volz RG. Efficacy of a topical antibiotic irrigant in decreasing or eliminating bacterial contamination in surgical wounds. Clin Orthop Relat Res. 1984;184:114-7. 10. Dirschl DR, Wilson FC. Topical antibiotic irrigation in the prophylaxis of operative wound infections in orthopedic surgery. Orthop Clin North Am. 1991; 22:419-26.




11. Lord JW Jr. Intraoperative antibiotic wound irrigation. Surg Gynecol Obstet. 1983;157:357-61. 12. Maguire WB. The use of antibiotics, locally and systemically, in orthopedic surgery. Med J Aust. 1964;52:412-4. 13. Scherr DD, Dodd TA. In vitro bacteriological evaluation of the effectiveness of antimicrobial irrigating solutions. J Bone Joint Surg Am. 1976;58:119-22. 14. Rosenstein BD, Wilson FC, Funderburk CH. The use of bacitracin irrigation to prevent infection in postoperative skeletal wounds. An experimental study. J Bone Joint Surg Am. 1989;71:427-30. 15. Bhandari M, Adili A, Schemitsch EH. The efficacy of low-pressure lavage with different irrigating solutions to remove adherent bacteria from bone. J Bone Joint Surg Am. 2001;83:412-9. 16. Brown LL, Shelton HT, Bornside GH, Cohn I Jr. Evaluation of wound irrigation by pulsatile jet and conventional methods. Ann Surg. 1978;187:170-3. 17. Bhandari M, Thompson K, Adili A, Shaughnessy SG. High and low pressure irrigation in contaminated wounds with exposed bone. Int J Surg Investig. 2000;2:179-82.


23. Park SH, Silva M, Bahk WJ, McKellop H, Lieberman JR. Effect of repeated irrigation and debridement on fracture healing in an animal model. J Orthop Res. 2002;20:1197-204. 24. Bhandari M, Schemitsch EH. High-pressure irrigation increases adipocyte-like cells at the expense of osteoblasts in vitro. J Bone Joint Surg Br. 2002;84: 1054-61. 25. Dirschl DR, Duff GP, Dahners LE, Edin M, Rahn BA, Miclau T. High pressure pulsatile lavage irrigation of intraarticular fractures: effects on fracture healing. J Orthop Trauma. 1998;12:460-3. 26. Caprise PA Jr, Miclau T, Dahners LE, Dirschl DR. High-pressure pulsatile lavage irrigation of contaminated fractures: effects on fracture healing. J Orthop Res. 2002;20:1205-9. 27. Siragusa GR, Nawotka K, Spilman SD, Contag PR, Contag CH. Real-time monitoring of Escherichia coli O157:H7 adherence to beef carcass surface tissues with a bioluminescent reporter. Appl Environ Microbiol. 1999;65: 1738-45.

18. Keblish DJ, DeMaio M. Early pulsatile lavage for the decontamination of combat wounds: historical review and point proposal. Mil Med. 1998;163:844-6.

28. Rocchetta HL, Boylan CJ, Foley JW, Iversen PW, LeTourneau DL, McMillian CL, Contag PR, Jenkins DE, Parr TR Jr. Validation of a noninvasive, real-time imaging technology using bioluminescent Escherichia coli in the neutropenic mouse thigh model of infection. Antimicrob Agents Chemother. 2001;45:129-37.

19. Pronchik D, Barber C, Rittenhouse S. Low- versus high-pressure irrigation techniques in Staphylococcus aureus-inoculated wounds. Am J Emerg Med. 1999;17:121-4.

29. Kadurugamuwa JL, Sin L, Albert E, Yu J, Francis K, DeBoer M, Rubin M, Bellinger-Kawahara C, Parr TR Jr, Contag PR. Direct continuous method for monitoring biofilm infection in a mouse model. Infect Immun. 2003;71:882-90.

20. Wheeler CB, Rodeheaver GT, Thacker JG, Edgerton MT, Edilich RF. Side-effects of high pressure irrigation. Surg Gynecol Obstet. 1976;143;775-8.

30. Hamer ML, Robson MC, Krizek TJ, Southwick WO. Quantitative bacterial analysis of comparative wound irrigations. Ann Surg. 1975;181:819-22.

21. Hassinger SM, Harding G, Wongworawat MD. High-pressure pulsatile lavage propagates bacteria into soft tissue. Clin Orthop Relat Res. 2005;439:27-31.

31. Gustilo RB, Anderson JT. Prevention of infection in the treatment of one thousand and twenty-five open fractures of long bones: retrospective and prospective analyses. J Bone Joint Surg Am. 1976;58:453-8.

22. Bhandari M, Schemitsch EH, Adili A, Lachowski RJ, Shaughnessy SG. High and low pressure pulsatile lavage of contaminated tibial fractures: an in vitro study of bacterial adherence and bone damage. J Orthop Trauma. 1999;13: 526-33.

32. Gainor BJ, Hockman DE, Anglen JO, Christensen G, Simpson WA. Benzalkonium chloride: a potential disinfecting irrigation solution. J Orthop Trauma. 1997;11:121-5.
