Abstract
The purpose of this study was to evaluate the effects on health and kidney function of local implantation of commercial gentamicin-impregnated collagen sponges. Four healthy dogs were submitted to local surgical implantation of collagen impregnated sponges. Follow-up with serial physical examinations and measurements of serum creatinine, urea nitrogen, and gentamicin were performed for 7 d. There were no adverse reactions, or changes in measurements of kidney function.
Résumé
Étude clinique de l’implantation locale d’éponges de collagène imprégnées de gentamicine chez les chiens. Le but de la présente étude consistait à évaluer les effets sur la santé et la fonction rénale de l’implantation locale d’éponges de collagène commerciales imprégnées de gentamicine. Quatre chiens en santé ont subi une implantation chirurgicale locale d’éponges imprégnées de collagène. Un suivi avec des examens physiques et des mesures de la créatinine sérique, de l’azote uréique et de la gentamicine a été effectué pendant 7 jours. Il n’y a eu aucun effet indésirable ni changements dans les mesures de la fonction rénale.
(Traduit par Isabelle Vallières)
Introduction
Bone or joint infections are serious complications that may occur after open fractures, or bone or joint surgery (1,2). If initial treatment is not effective, the condition can become chronic and difficult to eradicate (3). Treatment for osteomyelitis involves aggressive local and systemic measures that include prolonged local and/or systemic antibiotic use and often revision surgery (3). Serious side effects may be incurred through the long-term systemic use of antibiotics at high doses. Also, due to the reduced vascular supply the concentration of systemic antibiotics might be decreased at the site of infection. The delivery and maintenance of therapeutic levels of antibiotic at the site of infection can be achieved by using implants or carriers that release antibiotics locally; these have significantly improved osteomyelitis treatment (2)
Many local antibiotic releasing systems have been developed in recent years (1). Currently, polymethylmethacrylate (PMMA), collagen, apatite-wollastonite glass ceramic blocks, hydroxyapatite blocks, polylactide/polyglycolide implants, and polylactate polymers are available for clinical use (3–6). The ideal carrier needs to be biocompatible, nontoxic and have well-documented structural, physical, biological, and immunological properties (7). One of the most commonly used antibiotics is gentamicin, a broad spectrum bactericide (7), which is a low-cost aminoglycoside effective against most bacteria implicated in bone infections. Its action is concentration dependent and there is very low bacterial resistance at high doses (7). Commercial gentamicin-impregnated collagen sponges have been used clinically and experimentally in humans, horses, and rabbits for successful treatment of bone and joint infections (1,6,8–10), but have not been investigated in dogs. Only 1 case report mentioned the use of such sponges in the treatment of septic arthritis in a dog (11). Furthermore, prolonged systemic therapy with gentamicin can have deleterious nephrotoxic effects in dogs (12–14). To our knowledge no clinical investigation has been conducted to identify toxic effects after local implantation of gentamicin-impregnated collagen sponges in dogs. The purpose of this study was to evaluate the effects of local implantation of commercial gentamicin impregnated collagen sponges in dogs. We hypothesized that local implantation of gentamicin impregnated collagen sponges affects canine renal parameters, namely blood urea and creatinine, urine specific gravity, and the presence of urine casts.
Materials and methods
This study was a prospective study on 4 clinically healthy dogs and was limited to a period of investigation of 1 wk after the surgical procedure.
Animals
The trial group consisted of 4 purebred beagles: 2 castrated males and 2 spayed females that were randomly selected from a colony of dogs at the Faculty of Veterinary Medicine in Saint-Hyacinthe, Quebec. The dogs were middle-aged and had an average weight of 11.0 kg. All dogs were considered healthy on the basis of a complete physical examination. The dogs had been regularly vaccinated and had not received systemic antibiotic at any time. The dogs were kept in individual cages for 1 d prior to the beginning of the study. They were fed twice daily with commercial maintenance dry dog food and water was available ad libitum. The dogs were fasted the night before the surgical procedure. All procedures were approved by the Ethics Committee on Animal Use of the Faculty of Veterinary Medicine, of Montreal University in accordance with Canadian standards.
Surgical procedure
Each dog received premedication with acepromazine (Atravet 10 mg/mL; Boehringer Ingelheim (Canada), Burlington, Ontario), 0.03 mg/kg body weight (BW), IM and hydromor-phone (Hydromorphone HP® 10; Sandoz Canada, Boucherville, Quebec), 0.05 mg/kg BW, IM 15 min before general anesthesia. Anesthesia was induced with thiopental (Thiotal® 20 mg/mL; Vétoquinol, Lavaltrie, Quebec), 10 mg/kg BW, IV. A relay with isoflurane (Isoflurane USP®; Pharmaceutical Partners of Canada, Richmond Hill, Ontario) (an average of 2%) was used to maintain anesthesia. Isotonic fluids (Lactated Ringer’s Solution USP; Baxter Corporation, Mississauga, Ontario) at a rate of 10 mL/kg BW per hour, IV were administered throughout the anesthesia.
The dogs were placed in left lateral recumbancy and the right femur was surgically prepared to allow a lateral approach. All surgeries were performed by the same experienced surgeon. A 5-cm lateral distal femur skin incision was made and blunt dissection of subcutaneous tissue and muscle was performed to obtain an implantation site in contact with the bone. A collagen sponge impregnated with gentamicin (Collatamp G®; Theramed Corporation, Mississauga, Ontario) was then placed directly in contact with the bone and folded several times to exactly match the size of the incision. These sponges are made of type I collagen [2.5 mg/cm2 impregnated with gentamicin sulphate (2 mg/cm2)]. A standard surgical closure was used and a protective Opsite Post op® bandage (Smith Nephew Medical, Hull, England) was placed over the surgical wound.
Postoperative care
Postoperative pain was assessed every 2 h the first day after surgery and hydromorphone (Sandoz Canada), 0.05 mg/kg BW, SC was given q8h for 3 d, then as needed (but none of the dogs required supplementary doses after the 3-day postoperative period). Each dog was given a complete examination and measurement of parameters such as heart rate, respiratory rate, temperature, and vomiting were taken twice daily. Serum urea and creatinine, and urine specific gravity were measured on day 0 (the day of the surgery) and days 1 to 7 after the surgery. Serum gentamicin concentration was measured and pain was estimated on days 0 to 7 (scored using a numerical rating scale: 0 = no pain, 1 = pain on touch of incision site, 2 = pain on touch of the incision site and intermittent lameness on the operated limb, 3 = pain on touch of the incision site and non-weight-bearing on the operated limb).
Serum urea and creatinine and urine specific gravity of the first urine of the day taken by cystocentesis were determined each morning. Analyses were performed at the analytical laboratory of the Faculty of Veterinary Medicine. A blood sample was also collected each morning and serum was separated by centrifugation at 2000 × g for 5 min. The sample was then immediately placed in a freezer at −72°C for storage pending the determination of the concentration of gentamicin by immunofluorescence. This analysis was performed by Abbott laboratories (TDx/TDxFLx Gentamicin assay).
Results
Heart rate, respiratory rate, and body temperature remained within normal limits for each dog and there was no change in the breathing patterns between pre- and postoperative examinations. Functional recovery after surgery was excellent. No dog showed any lameness, discharge, or discomfort at the surgical site after implantation of the sponges and there were no postoperative complications.
A daily evaluation of urea and creatinine in blood (Table 1) revealed that the urea and serum creatinine remained within the normal limits throughout the 7-day postoperative period (Table 1).
Table 1.
Changes in serum concentrations of urea, creatinine, and gentamicin and in urine specific gravity following implantation of a collagen sponge with gentamicin
Day 0 | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | Day 7 | |
---|---|---|---|---|---|---|---|---|
Urea (mmol/L) | 6.65 ± 6.55a | 4.78 ± 2.78 | 3.90 ± 0.27 | 3.34 ± 0.36 | 4.05 ± 0.89 | 4.20 ± 0.65 | 4.75 ± 1.00 | 5.84 ± 0.37 |
Creatinine (μmol/L) | 101.5 ± 68.45 | 85.9 ± 37.28 | 67.75 ± 7.13 | 70.25 ± 6.85 | 70.0 ± 4.83 | 71.0 ± 6.68 | 69.25 ± 4.34 | 71.66 ± 4.04 |
Urine specific gravity | 1.036 ± 0.156 | 1.040 ± 0.191 | 1.043 ± 0.127 | 1.036 ± 0.130 | 1.040 ± 0.084 | 1.046 ± 0.140 | 1.048 ± 0.156 | 1.043 ± 0.125 |
Gentamicin (μg/L) | 0 ± 0 | 0.6 ± 0.74 | 0 ± 0 | 0 ± 0 | 0 ± 0 | 0 ± 0 | 0 ± 0 | 0 ± 0 |
± standard deviation.
The urine specific gravity remained essentially unchanged (Table 1). No casts were found on microscopic examination of the urine.
The daily serum concentration of gentamicin showed that the drug was detected at low levels on day 1 and was subsequently not detected.
Discussion
The results of our study suggest that the gentamicin- impregnated collagen sponges posed no risk to the kidney function of healthy dogs. These sponges have been used for a variety of infections in dogs, and in ophthalmic surgery or dentistry in humans (7). They have also been used in studies of the treatment of osteomyelitis in calves (8), septic arthritis in horses (1,9), and abdominal surgery in rats (10). Only 1 study reported the use of collagen sponges impregnated with gentamicin in dogs (11) and no study has highlighted the lack of systemic toxicity of the implants for this species.
Collagen presents several advantages as a carrier over the widely used PMMA (7) in the form of cement or rosary beads. Polymethylmethacrylate allows a gradual and prolonged local release of antibiotics, but has several disadvantages, in particular the need to remove the foreign material under general anesthesia (1,4,5,7,8). Among other antibiotic carriers with bioresorbable properties [hydroxyapatite, plaster of Paris, polyanhydrates, hyaluronan, polylactide-polyglycolides, momoolein-water liquid crystalline gels (1,5)], none is as biocompatible as collagen, which is a natural endogenous substance that makes up 95% of organic bone matrix and is the main component of cartilage and tissue connection. The collagen sponges used in our study are made of type I collagen purified from bovine Achilles tendon (15). The gradual degradation by natural collagenases allows gradual and continuous release of the antibiotic in the sponge. Collagen is also hemostatic, soft, and malleable, permitting adjustment to the size of the wound (11). It appears that the collagen interacts with platelets, fibroblasts, and macrophages, which has a positive influence on wound healing (2,15).
Gentamicin has the interesting property of diffusing into biofilms at high concentration. Co-lyophilization fixation of the antibiotic in the web of collagen allows uniform incorporation of the molecule in the matrix of the sponge and provides a uniform dose per cm2. Systemic injectable gentamicin sulfate has a high solubility in water (> 1 g/mL) but a low solubility in organic solvents (0.678 mg/mL in chloroform) which makes its tissue penetration very low. However, tissue accumulation can be found in the renal cortex and the endolymph of the inner ear, which may be the cause of nephro- and oto-toxicity (7,14,16).
Gentamicin has a “concentration-dependent” mode of action, which means that the bactericidal effect increases as the concentration of antibiotic increases. Gentamicin also has a “post-antibiotic effect” which means that inhibition of bacterial growth persists even when the antibiotic concentration falls below the minimum inhibitory concentration (MIC) (7). The known toxic concentration of gentamicin is 10 to 12 μg/mL (2), but is less in the presence of subclinical renal disease (13). In this study, no renal toxicity was evident with the use of the sponges in healthy dogs. However, no information is available on the local distribution of the antibiotic in the surgical area or the safe use of these sponges in hypotensive, renal disorders or traumatized patients.
The release of gentamicin from the sponges used in this study has never been studied in dogs. Several in vivo and in vitro studies in humans show that dissemination of gentamicin from the sponge is complete and the maximum threshold is reached quickly (2,9,15). Although our study involved only a few animals, the use of the sponges impregnated with gentamicin did not result in any significant level in serum and therefore had no clinical consequence.
According to the manufacturer’s data, serum peak gentamicin concentration occurs in the first hours following the introduction of the implant and therefore it could not be detected in our protocol. Further studies are needed to assess the local concentrations of the antibiotic, the diffusion pattern, the local tissue reactions to the presence of the implant, the period in which the concentration is higher than the recommended values. The collagen sponges impregnated with gentamicin have the disadvantage compared to PMMA of adding no solidity to the fracture site and not allowing filling of bone defects or voids. Moreover, 1 sponge was implanted in our experimental model. It would be interesting to know the influence of local and systemic application of several sponges.
Other study limitations were low number of animals that did not allow for meaningful statistics and monitoring was for only 7 d. Furthermore, no functional study of the kidneys (scintigraphy, biopsy) or the local gentamicin concentration (diffusion, transmission time, duration of concentration above the MIC) was performed. Further studies are required in order to certify the safety of these sponges in clinically and pathologically affected dogs.
Acknowledgments
The authors thank Theramed for providing the Collatamp G® sponges. We also thank Ms Rebecca de Arburn Parent for help with English translation. CVJ
Footnotes
Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office (hbroughton@cvma-acmv.org) for additional copies or permission to use this material elsewhere.
References
- 1.Cruz AM, Rubio-Martinez L, Dowling T. New antimicrobials, systemic distribution, and local methods of antimicrobial delivery in horses. Vet Clin North Am Equine Pract. 2006;22:297–322. vii–viii. doi: 10.1016/j.cveq.2006.03.006. [DOI] [PubMed] [Google Scholar]
- 2.Ruszczak Z, Friess W. Collagen as a carrier for on-site delivery of antibacterial drugs. Adv Drug Deliv Rev. 2003;55:1679–1698. doi: 10.1016/j.addr.2003.08.007. [DOI] [PubMed] [Google Scholar]
- 3.Seber S, Gunal I, Gokturk E. Antibiotic-impregnated xenografts in the treatment of chronic osteomyelitic cavities. Seven cases followed for 3 to 5 years. Int Orthop. 1998;22:197–199. doi: 10.1007/s002640050241. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Jenny G. Agents anti-microbiens dans le traitement local de l’infection osté-articulaire Antimicrobial local care in bone and joint infections. Orthopedie Traumatologie. 1994;4:109–111. [Google Scholar]
- 5.Ouedraogo M, Semdé R, Somé IT, et al. Monoolein-water liquid crystalline gels of gentamicin as bioresorbable implants for the local treatment of chronic osteomyelitis: In vitro characterization. Drug Dev Ind Pharm. 2008;34:753–760. doi: 10.1080/03639040801926287. [DOI] [PubMed] [Google Scholar]
- 6.Swieringa AJ, Goosen JH, Jansman FG, Tulp NJ. In vivo pharmacokinetics of a gentamicin-loaded collagen sponge in acute periprosthetic infection: Serum values in 19 patients. Acta Orthop. 2008;79:637–642. doi: 10.1080/17453670810016650. [DOI] [PubMed] [Google Scholar]
- 7.Ruszczak Z. Effect of collagen matrices on dermal wound healing. Adv Drug Deliv Rev. 2003;55:1595–1611. doi: 10.1016/j.addr.2003.08.003. [DOI] [PubMed] [Google Scholar]
- 8.Hirsbrunner G, Steiner A. Treatment of infectious arthritis of the radio-carpal joint of cattle with gentamicin-impregnated collagen sponges. Vet Rec. 1998;142:399–402. doi: 10.1136/vr.142.15.399. [DOI] [PubMed] [Google Scholar]
- 9.Summerhays GE. Treatment of traumatically induced synovial sepsis in horses with gentamicin-impregnated collagen sponges. Vet Rec. 2000;147:184–188. doi: 10.1136/vr.147.7.184. [DOI] [PubMed] [Google Scholar]
- 10.Vaneerdeweg W, Bresseleers T, Du Jardin P, et al. Comparison between plain and gentamicin containing collagen sponges in infected peritoneal cavity in rats. Eur J Surg. 1998;164:617–621. doi: 10.1080/110241598750005723. [DOI] [PubMed] [Google Scholar]
- 11.Owen MR, Moores AP, Coe RJ. Management of MRSA septic arthritis in a dog using a gentamicin-impregnated collagen sponge. J Small Anim Pract. 2004;45:609–612. doi: 10.1111/j.1748-5827.2004.tb00183.x. [DOI] [PubMed] [Google Scholar]
- 12.Albarellos G, Montoya L, Ambros L, Kreil V, Hallu R, Rebuelto M. Multiple once-daily dose pharmacokinetics and renal safety of gentamicin in dogs. J Vet Pharmacol Ther. 2004;27:21–25. doi: 10.1046/j.0140-7783.2003.00545.x. [DOI] [PubMed] [Google Scholar]
- 13.Frazier DL, Dix LP, Bowman KF, Thompson C, Riviere JE. Increased gentamicin nephrotoxicity in normal and diseased dogs administered identical serum drug concentration profiles: Increased sensitivity in subclinical renal dysfunction. J Pharmacol Exp Ther. 1986;239:946–951. [PubMed] [Google Scholar]
- 14.Servais H, Ortiz A, Devuyst O, Denamur S, Tulkens PM, Mingeot-Leclercq MP. Renal cell apoptosis induced by nephrotoxic drugs: Cellular and molecular mechanisms and potential approaches to modulation. Apoptosis. 2008;13:11–32. doi: 10.1007/s10495-007-0151-z. [DOI] [PubMed] [Google Scholar]
- 15.Stemberger A, Grimm H, Bader F, Rahn HD, Ascherl R. Local treatment of bone and soft tissue infections with the collagen-gentamicin sponge. Eur J Surg Suppl. 1997:17–26. [PubMed] [Google Scholar]
- 16.Zhou Y, Vaidya VS, Brown RP, et al. Comparison of kidney injury molecule-1 and other nephrotoxicity biomarkers in urine and kidney following acute exposure to gentamicin, mercury, and chromium. Toxicol Sci. 2008;101:159–170. doi: 10.1093/toxsci/kfm260. [DOI] [PMC free article] [PubMed] [Google Scholar]