Q U I N T E S S E N C E I N T E R N AT I O N A L
ORAL SURGERY
Bernhard Pommer
Decontamination of autogenous bone grafts: Systematic literature review and evidence-based proposal of a protocol Bernhard Pommer, DDS, PhD1/Apostolos Georgopoulos, MD, PhD2/Gabriella Dvorak, DDS, MD, PhD3/Christian Ulm, DDS, MD, PhD 3 There is a lack of consensus guidelines for the decontamination of autogenous bone grafts after exposure to a nonsterile environment during graft contouring, intraoral bone harvesting, or when inadvertently dropped off the sterile field. Transplantation of contaminated bone may cause infectious complications or even augmentation failure. When selecting the antimicrobial agent of choice to treat harvested bone for transplantation purposes, focus should be placed on the safety of the agent towards bone and osteoprogenitor cells, maximum elimination of targeted pathogens that directly affect bone tissue, and a short exposure time. In systematically reviewing study results on the degree of bone graft contamination and the effects of decontamination methods, a protocol
for decontamination of autogenous bone grafts is proposed. Among various decontamination agents investigated, 1% chlorhexidine proved highly effective (mean reduction of bacterial colony-forming units compared to saline solution: 99.97%). Minimum contact time required is 15 seconds and cell proliferation can be observed up to 30 seconds of exposure. The proposed decontamination protocol (1% chlorhexidine for 15 seconds) seems to represent a reasonable compromise in reference to sterility and cell viability. Comparative effectiveness research, however, is needed before clinical recommendations may be posed. (Quintessence Int 2014;45:145–150; doi: 10.3290/j.qi.a31011)
Key words: agent concentration, bone transplants, exposure time, graft decontamination, graft failure, graft infection
Despite recent advances in bone substitute technology, the use of autologous bone grafts is still considered the gold standard in reconstructive bone surgery1 because of their osteogenic, osteoinductive, osteoconductive, and nonimmunogenic properties. 1
Associate Professor, Department of Oral Surgery, Bernhard Gottlieb University Clinic of Dentistry, Vienna Medical University, Austria; and Academy for Oral Implantology, Vienna, Austria.
2
Associate Professor, Competence Center for Oral Microbiology, Bernhard Gottlieb University Clinic of Dentistry, Vienna Medical University, Austria.
3
Associate Professor, Department of Oral Surgery, Bernhard Gottlieb University Clinic of Dentistry, Vienna Medical University, Austria.
Correspondence: PD Dr Bernhard Pommer, Academy for Oral Implantology, Lazarettgasse 19/DG, A-1090 Vienna, Austria. Email: pommer@ implantatakademie.at
VOLUME 45 • NUMBER 2 • FEBRUARY 2014
The downsides of harvesting autologous bone, however, involve donor site morbidity, limited availability, as well as susceptibility to microbial contamination.2 Bone blocks require contouring prior to transplantation in most cases and may come in contact with nonsterile surfaces not only during graft manipulation but also when inadvertently dropped off the sterile field during surgery.3 Bone collected from the oral cavity is particularly susceptible to contamination, because viable microorganisms may reach up to 109 colonyforming units (CFU)/ml saliva (Fig 1).4 Transplantation of contaminated bone may cause infectious complications or even augmentation failure. Reported rates of
145
Q U I N T E S S E N C E I N T E R N AT I O N A L Pommer et al
tive in reducing bacterial contamination,14 there is a need to know what irrigation substance and decontamination protocol to use. The focused question addressed in the present literature survey is whether a protocol for decontamination of autogenous bone grafts can be proposed based on study results on the degree of bone graft contamination and the effects of decontamination methods. The ideal disinfectant would destroy contaminating organisms while maintaining the viability of bone and osteoprogenitor cells.12 Fig 1 Intraoral bone harvesting (using a bone filter attached to the suction system) associated with the potential of microbial graft contamination.
infection following graft contamination range from 5% to 50%.5 Several studies have determined the degree of bone graft contamination both quantitatively and qualitatively (Table 1). While bone grafts dropped on the operating room floor are contaminated in 60% to 96% of cases,12 intraoral bone samples yield viable microorganisms in up to 100%.8 Although bacterial count and spectrum are influenced by various factors and the composition of the bacterial flora varies from person to person,10 the most frequently identified bacteria belong to Streptococcus, Bacteroides, Peptococcus, Peptostreptococcus, or Veillonella species. Microorganisms relevant to bone graft infection involve Actinomyces odontolyticus, Clostridium bifermentans, Eubacterium species, Fusobacterium nucleatum, Peptostreptococcus species, Prevotella oralis, Prevotella intermedia, Staphylococcus species, and Veillonella species.6,7,13 There is a lack of consensus guidelines for the decontamination of native, living, autogenous bone after exposure to a nonsterile environment.3 Intraoperative management of contaminated bone grafts has received some attention in literature; however, the effect of sterilization on the graft material should not jeopardize the successful outcome of the reconstruction. As antibiotic prophylaxis has shown to be ineffec-
146
Systematic literature search Studies investigating either bacterial contamination or cell viability of autologous bone grafts prior to and/or following application of decontamination agents were considered eligible for inclusion. An electronic Medline search of English literature was conducted on January 1st 2013 using the keywords “bone graft decontamination”, “bone graft bacterial contamination”, “autogenous bone microbial analysis”, “disinfecting agents bacteria”, and “bone irrigation solutions” and was supplemented by hand-searching the references of relevant articles and review publications.4 Two reviewers (BP and GD) independently screened the titles and abstracts of the electronic search results (539 publications after exclusion of duplicates). Full texts of all papers that were considered eligible for inclusion by one or both of the reviewers were obtained for further assessment. Disagreement was resolved by consensus. Twenty-six studies5-30 constituted the final selection, eight of which16,21,22,23,26,27,29,30 were identified via hand-searching.
Decontamination agents The ability of various decontamination agents to remove adherent bacteria from bone surfaces (Table 2) has been previously reported.2,15,16 Compared to saline solution (747 ± 52.4 CFU), the residual number of bacterial CFUs following irrigation with 1% bacitracin was 630 ± 10.5 CFU (15.66% mean reduction), 319 ± 13.7 CFU with 1% ethanol (57.30% mean reduction), 35 ± 5.8 CFU with 1% liquid soap (95.31% mean reduction), 0.67 ± 0.33 CFU with 1% povidone-iodine (99.91% mean reduction), and 0.33 ± 0.33 CFU for 1%
VOLUME 45 • NUMBER 2 • FEBRUARY 2014
Q U I N T E S S E N C E I N T E R N AT I O N A L Pommer et al
Table 1
Microorganisms identified in studies on bone graft contamination
Actinomyces israelii Actinomyces meyeri Actinomyces naeslundii Actinomyces odontolyticus Aerococcus viridans Bacteroides species Bifidobacterium species Candida albicans Capnocytophaga species Clostridium bifermentans Clostridium tyrobutyricum Corynebacterium xerosis Dermacoccus nishinomyaensis Eggerthella lenta Eikenella corrodens Enterococcus faecalis Facklamia hominis Fusobacterium nucleatum Gemella haemolysans Gemella morbillorum Haemophilus influenzae Haemophilus parainfluenzae Haemophilus segnis Kaistella koreensis Klebsiella pneumoniae Kocuria kristinae Lactococcus lactis Leptotrichia buccalis Leuconostoc species Micrococcus luteus Micrococcus lylae Moraxella species Neisseria species Peptococcus species Peptostreptococcus species Porphyromonas endodontalis Prevotella bivia Prevotella buccalis Prevotella denticola Prevotella intermedia Prevotella melaninogenica Prevotella oralis Propionibacterium acnes Propionibacterium propionicum Pseudomonas aeruginosa Rothia dentocariosa Staphylococcus aureus Staphylococcus capitis Staphylococcus chromogenes Staphylococcus epidermidis Staphylococcus saccharolyticus Staphylococcus saprophyticus Staphylococcus sciuri Stomatococcus mucilaginosus Streptococcus acidominimus Streptococcus adjacens Streptococcus alactolyticus Streptococcus anginosus Streptococcus constellatus Streptococcus gordonii Streptococcus intermedius Streptococcus mitis Streptococcus mutans Streptococcus oralis Streptococcus parasanguis Streptococcus pneumoniae Streptococcus pyogenes Streptococcus salivarius Streptococcus sanguis Streptococcus vestibularis Terrahaemophilus aromaticivorans Veillonella species Wolinella species
Young et al6
Young et al7
x x
x x
Kürkçü et al8 x x x x
Kuttenberger Lambrecht et al9 et al10 x x x x x x x x
Sivolella et al11
x x x x
x
x x x x x
x
x
x
x
x x x
x x
x x
x
x
x x x
x x
x x x
x x x
x x x
x x x
x x x
x x
x
x x x
x x x x x x
x
x
x x x x x
x x x x
x
x x
x x
x x x x x
x x x x x
x x
x x
x x
x x x
x x x x x x
x x
x
x
x
x
x
x x
VOLUME 45 • NUMBER 2 • FEBRUARY 2014
x x x x x
x x x x x x x x x
x
x x x
x
x x
147
Q U I N T E S S E N C E I N T E R N AT I O N A L Pommer et al
Table 2
Percentage reduction of bacterial colony-forming units (CFUs) in bone grafts using various decontamination agents5,11,17,18
Decontamination agent
CFU reduction
Chlorhexidine
99.97%
Povidone-iodine
99.91%
Rifamycin
99.23%
Clindamycin
96.73%
Liquid soap
95.31%
Tetracycline
87.64%
Ethanol
57.30%
Bacitracin
15.66%
Decontamination protocol Effectiveness of bone graft decontamination may vary with different contact time and various agent concentrations.4 A safe, yet effective concentration of the drug should be established to prevent osseous tissue damage.24 In vitro studies revealed a significantly greater decrease in proportion of alkaline-phosphatase-positive
148
cells after exposure to 4% chlorhexidine-gluconate solution compared to the 1% irrigating solution.5 Trypan blue staining to determine cell viability following 2% chlorhexidine decontamination revealed severely reduced cell counts,25 while no significant reduction of osteoblastic outgrowth (97% to 100%) could be observed with concentrations up to 1% (Fig 2).24 Lower concentrations, on the other hand, resulted in incomplete sterilization: 0.1% and 0.2% chlorhexidine decontaminated only 67%9 and 86%18 of samples, respectively. Mean reduction of CFU using 0.12% chlorhexidine was 52.13%17 compared to 99.97% with 1% chlorhexidine.5 A
100 Osteoblastic positive growth (%)
chlorhexidine gluconate (99.97% mean reduction).5 This compares to a bacterial load reduction by antibiotics of 87.64% using tetracycline,17 96.73% using clindamycin,18 and 99.23% using rifamycin.11 There seems to be consensus in literature that povidoneiodine and chlorhexidine are the most effective decontamination agents.19 Compared to chlorhexidine, however, povidone-iodine did not prove to be effective against all bacteria relevant for decontamination of intraoral bone grafts12 nor against all organisms on bone grafts that had fallen onto the floor of the surgical ward.20 In a comparison of three sterilization agents used in orthopedic surgery, povidone-iodine was least effective for decontamination.21 Moreover, povidoneiodine was found to have toxic effects against granulocytes and monocytes even at low concentrations, and to provoke attacks through secondary thrombosis in the vascular epithelium.22 Chlorhexidine, by contrast, did not show any significant effect on growth of human gingival fibroblasts at concentrations ≤ 5 μmol/l23 and is associated with less potential for cell toxicity.
80
60
40
20
0.2% 1.0%
0 15
30
45
Time (seconds) Fig 2 Reduction of osteoblastic positive growth (%) after exposure times of 15, 30 and 45 seconds using 0.2 % (blue curve) vs 1.0 % chlorhexidine solution (red curve).24
VOLUME 45 • NUMBER 2 • FEBRUARY 2014
Q U I N T E S S E N C E I N T E R N AT I O N A L Pommer et al
concentration of 1% seems to represent a reasonable compromise in reference to sterility and cell viability. Bone decontamination using chlorhexidine shows a time-dependent effect on osteoblasts and osteoclasts.5 While long-term exposure to concentrations of 0.0001% to 0.00001% has little effect on cell growth and has been shown to cause induction in the synthesis of alkaline phosphatase,26 cell death occurred within 2 minutes in cultures where osteoblasts were exposed to 0.12% to 2% chlorhexidine.5 In fact, dilutions of less than 0.002% would be necessary to preserve cell vitality when applied for 5 minutes or more.27 As cytotoxicity on human osteoblast cells increases with longer time exposure, minimum contact time should be respected.28 In vitro studies showed that the minimum exposure time required for 1% chlorhexidine to produce negative cultures of tested pathogens (Staphylococcus aureus, Enterococcus faecalis, Candida albicans, Porphyromonas endodontalis, Porphyromonas gingivalis, P intermedia) was 15 seconds and cell proliferation was observed up to 30 seconds of exposure.29,30
DISCUSSION The proposed decontamination protocol does not pose cytotoxic effects on osteoblast growth and differentiation and may thus safely be used in bone transplantation surgery. However, other antimicrobial agents or a combination of different antibiotics could possibly achieve more effective decontamination. As cytotoxicity seems to be dependent on agent concentration as well as exposure duration, further research is indicated to determine the most effective protocol to completely remove bacteria from bone grafts (or at least reduce them at the best possible rate) while still preserving the inherent osteogenic potential of autologous grafts. Scientific evidence to support the premise that viable bone cells contribute to the process of graft consolidation, however, is still limited.1 Furthermore, the correlation between bacterial load of the graft and complication rates during bone healing needs to be elucidated.13 Results of a case series suggest that microbial contamination may have an impact on osteogenesis in
VOLUME 45 • NUMBER 2 • FEBRUARY 2014
bone regeneration;28 however, pronounced volume loss and bone graft resorption could not yet be clearly associated to the presence of certain pathogens. To date, scientific evidence to support the premise that contaminated bone grafts hamper the process of graft consolidation is limited. To the best of the authors’ knowledge there is no scientific literature to date that investigated both residual bacterial load as well as osteogenic potential of decontaminated bone and correlated these findings to osteogenesis and healing capacity of the graft in a clinical setting.18 Furthermore, it is not completely understood what modifications of bone cells as well as the collagen matrix are induced by antimicrobial agents. It can thus not be ruled out that decontamination might have no (or even negative) effects on bone regeneration.4 Possible negative consequences of graft decontamination may involve increased risk of sequestration and a reduced amount of newly formed bone or delay in bone remodeling. Even though comparisons have to be drawn carefully, antibiotic-supplemented bone grafts have been used successfully in third molar extraction sockets31 as well as bone fractures32 without disturbing osteogenesis. In treating osteomyelitis and infections related to orthopedic prostheses, antimicrobial agents have been shown to provide a high degree of diffusion inside osseous tissue.24 Antimicrobial activity of chlorhexidine for up to 72 hours has been reported following mouth rinses,33 yet it is not known how long decontaminating effects will last in grafted bone. It has at least been demonstrated that autogenous bone soaked in disinfectants and grafted can carry the drug to the grafting site.34 When selecting the antimicrobial agent of choice to treat harvested bone for transplantation purposes, focus should be placed on the safety of the agent towards bone and osteoprogenitor cells, maximum elimination of targeted pathogens that directly affect bone tissue, and a short contact time.24 In reviewing the scientific literature it is plausible to propose that bone decontamination for grafting purposes using 1% chlorhexidine is feasible within a short exposure time of 15 to 30 seconds. Current in vitro evidence suggests that
149
Q U I N T E S S E N C E I N T E R N AT I O N A L Pommer et al
the proposed decontamination protocol may safely be used in bone transplantation surgery; however, comparative effectiveness research is needed before clinical recommendations may be posed.
REFERENCES 1. Rickert D, Slater JJ, Meijer HJ, Vissink A, Raghoebar GM. Maxillary sinus lift with solely autogenous bone compared to a combination of autogenous bone and growth factors or (solely) bone substitutes. A systematic review. Int J Oral Maxillofac Surg 2012;41:160–167. 2. Pommer B, Tepper G, Gahleitner A, Zechner W, Watzek G. New safety margins for chin bone harvesting based on the course of the mandibular incisive canal in CT. Clin Oral Implants Res 2008;19:1312–1316. 3. Kang L, Mermel LA, Trafton TG. What happens when autogenous bone drops out of the sterile field during orthopaedic trauma surgery. J Orthop Trauma 2008;22:430–431. 4. Tezulas E, Dilek OC. Decontamination of autogenous bone grafts collected from dental implant sites via osteotomy: a review. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:679–684. 5. 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 2001;83-A:412–419. 6. Young MP, Carter DH, Worthington H, Korachi M, Drucker DB. Microbial analysis of bone collected during implant surgery: a clinical and laboratory study. Clin Oral Implants Res 2001;12:95–103. 7. Young MP, Korachi M, Carter DH, Worthington HV, McCord JF, Drucker DB. The effects of an immediately pre-surgical chlorhexidine oral rinse on the bacterial contaminants of bone debris collected during dental implant surgery. Clin Oral Implants Res 2002;13:20–29. 8. Kürkçü M, Oz IA, Köksal F, Benlidayi ME, Güneşli A. Microbial analysis of the autogenous bone collected by bone filter during oral surgery: a clinical study. Int J Oral Maxillofac Surg 2005;63:1593–1598. 9. Kuttenberger JJ, Hardt N, Rutz T, Pfyffer GE. Bone collected with a bone collector during dental implant surgery. Mund Kiefer Gesichtschir 2005;9:18–23. 10. Lambrecht JT, Glaser B, Meyer J. Bacterial contamination of filtered intraoral bone chips. Int J Oral Maxillofac Surg 2006;35:996–1000. 11. Sivolella S, Berengo M, Scarin M, Mella F, Martinelli F. Autogenous particulate bone collected with a piezo-electric surgical device and bone trap: a microbiological and histomorphometric study. Arch Oral Biol 2006;51:883–891. 12. Yaman F, Unlü G, Atilgan S, Celik Y, Ozekinci T, Yaldiz M. Microbiologic and histologic assessment of intentional bacterial contamination of bone grafts. J Oral Maxillofac Surg 2007;65:1490–1494. 13. Glaser B, Hodel Y, Meyer J, Lambrecht JT. Bacterial contamination of bony particles from the bone collection trap. Schweiz Monatsschr Zahnmed 2004;114:337–341. 14. Blay A, Tunchel S, Sendyk WR. Viability of autogenous bone grafts obtained by using bone collectors: histological and microbiological study. Pesqui Odontol Bras 2003;17:234–240. 15. Anglen JO, Apostoles S, Christensen G, Gainor B. The efficacy of various irrigation solutions in removing slime-producing Staphylococcus. J Orthop Trauma 1994;8:390–396.
150
16. Moussa FW, Gainor BJ, Anglen JO, Christensen G, Simpson WA. Disinfecting agents for removing adherent bacteria from orthopaedic hardware. Clin Orthop Relat Res 1996;329:255–262. 17. Etcheson AW, Miley DD, Gillespie MJ. Osseous coagulum collected in bone traps: potential for bacterial contamination and methods for decontamination. J Oral Implantol 2007;33:109–115. 18. Tezulas E, Dilek OC, Topcuoglu N, Kulekci G. Decontamination of autogenous bone grafts collected during dental implant site preparation: a pilot study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;107:656–660. 19. Bruce B, Sheibani-Rad S, Appleyard D, Calfee RP, Reinert SE, Chapin KC, DiGiovanni CW. Are dropped osteoarticular bone fragments safely reimplantable in vivo? J Bone Joint Surg Am 2011;93:430–439. 20. Goebel ME, Drez Jr D, Heck SB, Stoma MK. Contaminated rabbit patellar tendon grafts. In vivo analysis of disinfecting methods. Am J Sports Med 1994;22:387–391. 21. Molina ME, Nonweiller DE, Evans JA, Delee JC. Contaminated anterior cruciate ligament grafts: the efficacy of 3 sterilization agents. Arthroscopy 2000;16:373–378. 22. Severyns AM, Lejeune A, Rocoux G, Lejeune G. Non-toxic antiseptic irrigation with chlorhexidine in experimental revascularization in the rat. J Hosp Infect 1991;17:197–206. 23. Dogan S, Günay H, Leyhausen G, Geurtsen W. Effects of low-concentrated chlorhexidine on growth of Streptococcus sobrinus and primary human gingival fibroblasts. Clin Oral Investig 2003;7:212–216. 24. Verdugo F, Sáez-Rosón A, Uribarri A, et al. Bone microbial decontamination agents in osseous grafting: an in vitro study with fresh human explants. J Periodontol 2011;82:863–871. 25. Bauer J, Liu RW, Kean TJ, Dennis JE, Petersilge W, Gilmore A. A comparison of five treatment protocols for contaminated bone grafts in reference to sterility and cell viability. J Bone Joint Surg 2001;93:439–444. 26. Cabral CT, Fernandes MH. In vitro comparison of chlorhexidine and povidoneiodine on the long-term proliferation and functional activity of human alveolar bone cells. Clin Oral Investig 2007;11:155–164. 27. Patel P, Ide M, Coward P, Di Silvio L. The effect of a commercially available chlorhexidine mouthwash product on human osteoblast cells. Eur J Prosthodont Restor Dent 2006;14:67–72. 28. Verdugo F, Castillo A, Moragues MD, Pontón J. Bone microbial contamination influences autogenous grafting in sinus augmentation. J Periodontol 2009;80:1355–1364. 29. Gomes BP, Ferraz CC, Vianna ME, Berber VB, Teixeira FB, Souza-Filho FJ. In vitro antimicrobial activity of several concentrations of sodium hypochlorite and chlorhexidine gluconate in the elimination of Enterococcus faecalis. Int Endod J 2001;34:424–428. 30. Vianna ME, Gomes BP, Berber VB, Zaia AA, Ferraz CC, de Souza-Filho FJ. In vitro evaluation of the antimicrobial activity of chlorhexidine and sodium hypochlorite. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97:79–84. 31. Petri 3rd WH, Wilson TM. Clinical evaluation of antibiotic-supplemented bone allograft. J Oral Maxillofac Surg 1993;51:982–985. 32. Petri 3rd WH, Schaberg SJ. The effects of antibiotic-supplemented bone allografts on contaminated, partially avulsive fractures of the canine ulna. J Oral Maxillofac Surg 1984;42:699–704. 33. White RR, Hays GL, Janer LR. Residual antimicrobial activity after canal irrigation with chlorhexidine. J Endod 1997;23:229–231. 34. Witsø E, Persen L, Løseth K, Benum P, Bergh K. Cancellous bone as an antibiotic carrier. Acta Orthop Scand 2000;71:80–84.
VOLUME 45 • NUMBER 2 • FEBRUARY 2014
ORAL SURGERY
Bernhard Pommer
Decontamination of autogenous bone grafts: Systematic literature review and evidence-based proposal of a protocol Bernhard Pommer, DDS, PhD1/Apostolos Georgopoulos, MD, PhD2/Gabriella Dvorak, DDS, MD, PhD3/Christian Ulm, DDS, MD, PhD 3 There is a lack of consensus guidelines for the decontamination of autogenous bone grafts after exposure to a nonsterile environment during graft contouring, intraoral bone harvesting, or when inadvertently dropped off the sterile field. Transplantation of contaminated bone may cause infectious complications or even augmentation failure. When selecting the antimicrobial agent of choice to treat harvested bone for transplantation purposes, focus should be placed on the safety of the agent towards bone and osteoprogenitor cells, maximum elimination of targeted pathogens that directly affect bone tissue, and a short exposure time. In systematically reviewing study results on the degree of bone graft contamination and the effects of decontamination methods, a protocol
for decontamination of autogenous bone grafts is proposed. Among various decontamination agents investigated, 1% chlorhexidine proved highly effective (mean reduction of bacterial colony-forming units compared to saline solution: 99.97%). Minimum contact time required is 15 seconds and cell proliferation can be observed up to 30 seconds of exposure. The proposed decontamination protocol (1% chlorhexidine for 15 seconds) seems to represent a reasonable compromise in reference to sterility and cell viability. Comparative effectiveness research, however, is needed before clinical recommendations may be posed. (Quintessence Int 2014;45:145–150; doi: 10.3290/j.qi.a31011)
Key words: agent concentration, bone transplants, exposure time, graft decontamination, graft failure, graft infection
Despite recent advances in bone substitute technology, the use of autologous bone grafts is still considered the gold standard in reconstructive bone surgery1 because of their osteogenic, osteoinductive, osteoconductive, and nonimmunogenic properties. 1
Associate Professor, Department of Oral Surgery, Bernhard Gottlieb University Clinic of Dentistry, Vienna Medical University, Austria; and Academy for Oral Implantology, Vienna, Austria.
2
Associate Professor, Competence Center for Oral Microbiology, Bernhard Gottlieb University Clinic of Dentistry, Vienna Medical University, Austria.
3
Associate Professor, Department of Oral Surgery, Bernhard Gottlieb University Clinic of Dentistry, Vienna Medical University, Austria.
Correspondence: PD Dr Bernhard Pommer, Academy for Oral Implantology, Lazarettgasse 19/DG, A-1090 Vienna, Austria. Email: pommer@ implantatakademie.at
VOLUME 45 • NUMBER 2 • FEBRUARY 2014
The downsides of harvesting autologous bone, however, involve donor site morbidity, limited availability, as well as susceptibility to microbial contamination.2 Bone blocks require contouring prior to transplantation in most cases and may come in contact with nonsterile surfaces not only during graft manipulation but also when inadvertently dropped off the sterile field during surgery.3 Bone collected from the oral cavity is particularly susceptible to contamination, because viable microorganisms may reach up to 109 colonyforming units (CFU)/ml saliva (Fig 1).4 Transplantation of contaminated bone may cause infectious complications or even augmentation failure. Reported rates of
145
Q U I N T E S S E N C E I N T E R N AT I O N A L Pommer et al
tive in reducing bacterial contamination,14 there is a need to know what irrigation substance and decontamination protocol to use. The focused question addressed in the present literature survey is whether a protocol for decontamination of autogenous bone grafts can be proposed based on study results on the degree of bone graft contamination and the effects of decontamination methods. The ideal disinfectant would destroy contaminating organisms while maintaining the viability of bone and osteoprogenitor cells.12 Fig 1 Intraoral bone harvesting (using a bone filter attached to the suction system) associated with the potential of microbial graft contamination.
infection following graft contamination range from 5% to 50%.5 Several studies have determined the degree of bone graft contamination both quantitatively and qualitatively (Table 1). While bone grafts dropped on the operating room floor are contaminated in 60% to 96% of cases,12 intraoral bone samples yield viable microorganisms in up to 100%.8 Although bacterial count and spectrum are influenced by various factors and the composition of the bacterial flora varies from person to person,10 the most frequently identified bacteria belong to Streptococcus, Bacteroides, Peptococcus, Peptostreptococcus, or Veillonella species. Microorganisms relevant to bone graft infection involve Actinomyces odontolyticus, Clostridium bifermentans, Eubacterium species, Fusobacterium nucleatum, Peptostreptococcus species, Prevotella oralis, Prevotella intermedia, Staphylococcus species, and Veillonella species.6,7,13 There is a lack of consensus guidelines for the decontamination of native, living, autogenous bone after exposure to a nonsterile environment.3 Intraoperative management of contaminated bone grafts has received some attention in literature; however, the effect of sterilization on the graft material should not jeopardize the successful outcome of the reconstruction. As antibiotic prophylaxis has shown to be ineffec-
146
Systematic literature search Studies investigating either bacterial contamination or cell viability of autologous bone grafts prior to and/or following application of decontamination agents were considered eligible for inclusion. An electronic Medline search of English literature was conducted on January 1st 2013 using the keywords “bone graft decontamination”, “bone graft bacterial contamination”, “autogenous bone microbial analysis”, “disinfecting agents bacteria”, and “bone irrigation solutions” and was supplemented by hand-searching the references of relevant articles and review publications.4 Two reviewers (BP and GD) independently screened the titles and abstracts of the electronic search results (539 publications after exclusion of duplicates). Full texts of all papers that were considered eligible for inclusion by one or both of the reviewers were obtained for further assessment. Disagreement was resolved by consensus. Twenty-six studies5-30 constituted the final selection, eight of which16,21,22,23,26,27,29,30 were identified via hand-searching.
Decontamination agents The ability of various decontamination agents to remove adherent bacteria from bone surfaces (Table 2) has been previously reported.2,15,16 Compared to saline solution (747 ± 52.4 CFU), the residual number of bacterial CFUs following irrigation with 1% bacitracin was 630 ± 10.5 CFU (15.66% mean reduction), 319 ± 13.7 CFU with 1% ethanol (57.30% mean reduction), 35 ± 5.8 CFU with 1% liquid soap (95.31% mean reduction), 0.67 ± 0.33 CFU with 1% povidone-iodine (99.91% mean reduction), and 0.33 ± 0.33 CFU for 1%
VOLUME 45 • NUMBER 2 • FEBRUARY 2014
Q U I N T E S S E N C E I N T E R N AT I O N A L Pommer et al
Table 1
Microorganisms identified in studies on bone graft contamination
Actinomyces israelii Actinomyces meyeri Actinomyces naeslundii Actinomyces odontolyticus Aerococcus viridans Bacteroides species Bifidobacterium species Candida albicans Capnocytophaga species Clostridium bifermentans Clostridium tyrobutyricum Corynebacterium xerosis Dermacoccus nishinomyaensis Eggerthella lenta Eikenella corrodens Enterococcus faecalis Facklamia hominis Fusobacterium nucleatum Gemella haemolysans Gemella morbillorum Haemophilus influenzae Haemophilus parainfluenzae Haemophilus segnis Kaistella koreensis Klebsiella pneumoniae Kocuria kristinae Lactococcus lactis Leptotrichia buccalis Leuconostoc species Micrococcus luteus Micrococcus lylae Moraxella species Neisseria species Peptococcus species Peptostreptococcus species Porphyromonas endodontalis Prevotella bivia Prevotella buccalis Prevotella denticola Prevotella intermedia Prevotella melaninogenica Prevotella oralis Propionibacterium acnes Propionibacterium propionicum Pseudomonas aeruginosa Rothia dentocariosa Staphylococcus aureus Staphylococcus capitis Staphylococcus chromogenes Staphylococcus epidermidis Staphylococcus saccharolyticus Staphylococcus saprophyticus Staphylococcus sciuri Stomatococcus mucilaginosus Streptococcus acidominimus Streptococcus adjacens Streptococcus alactolyticus Streptococcus anginosus Streptococcus constellatus Streptococcus gordonii Streptococcus intermedius Streptococcus mitis Streptococcus mutans Streptococcus oralis Streptococcus parasanguis Streptococcus pneumoniae Streptococcus pyogenes Streptococcus salivarius Streptococcus sanguis Streptococcus vestibularis Terrahaemophilus aromaticivorans Veillonella species Wolinella species
Young et al6
Young et al7
x x
x x
Kürkçü et al8 x x x x
Kuttenberger Lambrecht et al9 et al10 x x x x x x x x
Sivolella et al11
x x x x
x
x x x x x
x
x
x
x
x x x
x x
x x
x
x
x x x
x x
x x x
x x x
x x x
x x x
x x x
x x
x
x x x
x x x x x x
x
x
x x x x x
x x x x
x
x x
x x
x x x x x
x x x x x
x x
x x
x x
x x x
x x x x x x
x x
x
x
x
x
x
x x
VOLUME 45 • NUMBER 2 • FEBRUARY 2014
x x x x x
x x x x x x x x x
x
x x x
x
x x
147
Q U I N T E S S E N C E I N T E R N AT I O N A L Pommer et al
Table 2
Percentage reduction of bacterial colony-forming units (CFUs) in bone grafts using various decontamination agents5,11,17,18
Decontamination agent
CFU reduction
Chlorhexidine
99.97%
Povidone-iodine
99.91%
Rifamycin
99.23%
Clindamycin
96.73%
Liquid soap
95.31%
Tetracycline
87.64%
Ethanol
57.30%
Bacitracin
15.66%
Decontamination protocol Effectiveness of bone graft decontamination may vary with different contact time and various agent concentrations.4 A safe, yet effective concentration of the drug should be established to prevent osseous tissue damage.24 In vitro studies revealed a significantly greater decrease in proportion of alkaline-phosphatase-positive
148
cells after exposure to 4% chlorhexidine-gluconate solution compared to the 1% irrigating solution.5 Trypan blue staining to determine cell viability following 2% chlorhexidine decontamination revealed severely reduced cell counts,25 while no significant reduction of osteoblastic outgrowth (97% to 100%) could be observed with concentrations up to 1% (Fig 2).24 Lower concentrations, on the other hand, resulted in incomplete sterilization: 0.1% and 0.2% chlorhexidine decontaminated only 67%9 and 86%18 of samples, respectively. Mean reduction of CFU using 0.12% chlorhexidine was 52.13%17 compared to 99.97% with 1% chlorhexidine.5 A
100 Osteoblastic positive growth (%)
chlorhexidine gluconate (99.97% mean reduction).5 This compares to a bacterial load reduction by antibiotics of 87.64% using tetracycline,17 96.73% using clindamycin,18 and 99.23% using rifamycin.11 There seems to be consensus in literature that povidoneiodine and chlorhexidine are the most effective decontamination agents.19 Compared to chlorhexidine, however, povidone-iodine did not prove to be effective against all bacteria relevant for decontamination of intraoral bone grafts12 nor against all organisms on bone grafts that had fallen onto the floor of the surgical ward.20 In a comparison of three sterilization agents used in orthopedic surgery, povidone-iodine was least effective for decontamination.21 Moreover, povidoneiodine was found to have toxic effects against granulocytes and monocytes even at low concentrations, and to provoke attacks through secondary thrombosis in the vascular epithelium.22 Chlorhexidine, by contrast, did not show any significant effect on growth of human gingival fibroblasts at concentrations ≤ 5 μmol/l23 and is associated with less potential for cell toxicity.
80
60
40
20
0.2% 1.0%
0 15
30
45
Time (seconds) Fig 2 Reduction of osteoblastic positive growth (%) after exposure times of 15, 30 and 45 seconds using 0.2 % (blue curve) vs 1.0 % chlorhexidine solution (red curve).24
VOLUME 45 • NUMBER 2 • FEBRUARY 2014
Q U I N T E S S E N C E I N T E R N AT I O N A L Pommer et al
concentration of 1% seems to represent a reasonable compromise in reference to sterility and cell viability. Bone decontamination using chlorhexidine shows a time-dependent effect on osteoblasts and osteoclasts.5 While long-term exposure to concentrations of 0.0001% to 0.00001% has little effect on cell growth and has been shown to cause induction in the synthesis of alkaline phosphatase,26 cell death occurred within 2 minutes in cultures where osteoblasts were exposed to 0.12% to 2% chlorhexidine.5 In fact, dilutions of less than 0.002% would be necessary to preserve cell vitality when applied for 5 minutes or more.27 As cytotoxicity on human osteoblast cells increases with longer time exposure, minimum contact time should be respected.28 In vitro studies showed that the minimum exposure time required for 1% chlorhexidine to produce negative cultures of tested pathogens (Staphylococcus aureus, Enterococcus faecalis, Candida albicans, Porphyromonas endodontalis, Porphyromonas gingivalis, P intermedia) was 15 seconds and cell proliferation was observed up to 30 seconds of exposure.29,30
DISCUSSION The proposed decontamination protocol does not pose cytotoxic effects on osteoblast growth and differentiation and may thus safely be used in bone transplantation surgery. However, other antimicrobial agents or a combination of different antibiotics could possibly achieve more effective decontamination. As cytotoxicity seems to be dependent on agent concentration as well as exposure duration, further research is indicated to determine the most effective protocol to completely remove bacteria from bone grafts (or at least reduce them at the best possible rate) while still preserving the inherent osteogenic potential of autologous grafts. Scientific evidence to support the premise that viable bone cells contribute to the process of graft consolidation, however, is still limited.1 Furthermore, the correlation between bacterial load of the graft and complication rates during bone healing needs to be elucidated.13 Results of a case series suggest that microbial contamination may have an impact on osteogenesis in
VOLUME 45 • NUMBER 2 • FEBRUARY 2014
bone regeneration;28 however, pronounced volume loss and bone graft resorption could not yet be clearly associated to the presence of certain pathogens. To date, scientific evidence to support the premise that contaminated bone grafts hamper the process of graft consolidation is limited. To the best of the authors’ knowledge there is no scientific literature to date that investigated both residual bacterial load as well as osteogenic potential of decontaminated bone and correlated these findings to osteogenesis and healing capacity of the graft in a clinical setting.18 Furthermore, it is not completely understood what modifications of bone cells as well as the collagen matrix are induced by antimicrobial agents. It can thus not be ruled out that decontamination might have no (or even negative) effects on bone regeneration.4 Possible negative consequences of graft decontamination may involve increased risk of sequestration and a reduced amount of newly formed bone or delay in bone remodeling. Even though comparisons have to be drawn carefully, antibiotic-supplemented bone grafts have been used successfully in third molar extraction sockets31 as well as bone fractures32 without disturbing osteogenesis. In treating osteomyelitis and infections related to orthopedic prostheses, antimicrobial agents have been shown to provide a high degree of diffusion inside osseous tissue.24 Antimicrobial activity of chlorhexidine for up to 72 hours has been reported following mouth rinses,33 yet it is not known how long decontaminating effects will last in grafted bone. It has at least been demonstrated that autogenous bone soaked in disinfectants and grafted can carry the drug to the grafting site.34 When selecting the antimicrobial agent of choice to treat harvested bone for transplantation purposes, focus should be placed on the safety of the agent towards bone and osteoprogenitor cells, maximum elimination of targeted pathogens that directly affect bone tissue, and a short contact time.24 In reviewing the scientific literature it is plausible to propose that bone decontamination for grafting purposes using 1% chlorhexidine is feasible within a short exposure time of 15 to 30 seconds. Current in vitro evidence suggests that
149
Q U I N T E S S E N C E I N T E R N AT I O N A L Pommer et al
the proposed decontamination protocol may safely be used in bone transplantation surgery; however, comparative effectiveness research is needed before clinical recommendations may be posed.
REFERENCES 1. Rickert D, Slater JJ, Meijer HJ, Vissink A, Raghoebar GM. Maxillary sinus lift with solely autogenous bone compared to a combination of autogenous bone and growth factors or (solely) bone substitutes. A systematic review. Int J Oral Maxillofac Surg 2012;41:160–167. 2. Pommer B, Tepper G, Gahleitner A, Zechner W, Watzek G. New safety margins for chin bone harvesting based on the course of the mandibular incisive canal in CT. Clin Oral Implants Res 2008;19:1312–1316. 3. Kang L, Mermel LA, Trafton TG. What happens when autogenous bone drops out of the sterile field during orthopaedic trauma surgery. J Orthop Trauma 2008;22:430–431. 4. Tezulas E, Dilek OC. Decontamination of autogenous bone grafts collected from dental implant sites via osteotomy: a review. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:679–684. 5. 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 2001;83-A:412–419. 6. Young MP, Carter DH, Worthington H, Korachi M, Drucker DB. Microbial analysis of bone collected during implant surgery: a clinical and laboratory study. Clin Oral Implants Res 2001;12:95–103. 7. Young MP, Korachi M, Carter DH, Worthington HV, McCord JF, Drucker DB. The effects of an immediately pre-surgical chlorhexidine oral rinse on the bacterial contaminants of bone debris collected during dental implant surgery. Clin Oral Implants Res 2002;13:20–29. 8. Kürkçü M, Oz IA, Köksal F, Benlidayi ME, Güneşli A. Microbial analysis of the autogenous bone collected by bone filter during oral surgery: a clinical study. Int J Oral Maxillofac Surg 2005;63:1593–1598. 9. Kuttenberger JJ, Hardt N, Rutz T, Pfyffer GE. Bone collected with a bone collector during dental implant surgery. Mund Kiefer Gesichtschir 2005;9:18–23. 10. Lambrecht JT, Glaser B, Meyer J. Bacterial contamination of filtered intraoral bone chips. Int J Oral Maxillofac Surg 2006;35:996–1000. 11. Sivolella S, Berengo M, Scarin M, Mella F, Martinelli F. Autogenous particulate bone collected with a piezo-electric surgical device and bone trap: a microbiological and histomorphometric study. Arch Oral Biol 2006;51:883–891. 12. Yaman F, Unlü G, Atilgan S, Celik Y, Ozekinci T, Yaldiz M. Microbiologic and histologic assessment of intentional bacterial contamination of bone grafts. J Oral Maxillofac Surg 2007;65:1490–1494. 13. Glaser B, Hodel Y, Meyer J, Lambrecht JT. Bacterial contamination of bony particles from the bone collection trap. Schweiz Monatsschr Zahnmed 2004;114:337–341. 14. Blay A, Tunchel S, Sendyk WR. Viability of autogenous bone grafts obtained by using bone collectors: histological and microbiological study. Pesqui Odontol Bras 2003;17:234–240. 15. Anglen JO, Apostoles S, Christensen G, Gainor B. The efficacy of various irrigation solutions in removing slime-producing Staphylococcus. J Orthop Trauma 1994;8:390–396.
150
16. Moussa FW, Gainor BJ, Anglen JO, Christensen G, Simpson WA. Disinfecting agents for removing adherent bacteria from orthopaedic hardware. Clin Orthop Relat Res 1996;329:255–262. 17. Etcheson AW, Miley DD, Gillespie MJ. Osseous coagulum collected in bone traps: potential for bacterial contamination and methods for decontamination. J Oral Implantol 2007;33:109–115. 18. Tezulas E, Dilek OC, Topcuoglu N, Kulekci G. Decontamination of autogenous bone grafts collected during dental implant site preparation: a pilot study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;107:656–660. 19. Bruce B, Sheibani-Rad S, Appleyard D, Calfee RP, Reinert SE, Chapin KC, DiGiovanni CW. Are dropped osteoarticular bone fragments safely reimplantable in vivo? J Bone Joint Surg Am 2011;93:430–439. 20. Goebel ME, Drez Jr D, Heck SB, Stoma MK. Contaminated rabbit patellar tendon grafts. In vivo analysis of disinfecting methods. Am J Sports Med 1994;22:387–391. 21. Molina ME, Nonweiller DE, Evans JA, Delee JC. Contaminated anterior cruciate ligament grafts: the efficacy of 3 sterilization agents. Arthroscopy 2000;16:373–378. 22. Severyns AM, Lejeune A, Rocoux G, Lejeune G. Non-toxic antiseptic irrigation with chlorhexidine in experimental revascularization in the rat. J Hosp Infect 1991;17:197–206. 23. Dogan S, Günay H, Leyhausen G, Geurtsen W. Effects of low-concentrated chlorhexidine on growth of Streptococcus sobrinus and primary human gingival fibroblasts. Clin Oral Investig 2003;7:212–216. 24. Verdugo F, Sáez-Rosón A, Uribarri A, et al. Bone microbial decontamination agents in osseous grafting: an in vitro study with fresh human explants. J Periodontol 2011;82:863–871. 25. Bauer J, Liu RW, Kean TJ, Dennis JE, Petersilge W, Gilmore A. A comparison of five treatment protocols for contaminated bone grafts in reference to sterility and cell viability. J Bone Joint Surg 2001;93:439–444. 26. Cabral CT, Fernandes MH. In vitro comparison of chlorhexidine and povidoneiodine on the long-term proliferation and functional activity of human alveolar bone cells. Clin Oral Investig 2007;11:155–164. 27. Patel P, Ide M, Coward P, Di Silvio L. The effect of a commercially available chlorhexidine mouthwash product on human osteoblast cells. Eur J Prosthodont Restor Dent 2006;14:67–72. 28. Verdugo F, Castillo A, Moragues MD, Pontón J. Bone microbial contamination influences autogenous grafting in sinus augmentation. J Periodontol 2009;80:1355–1364. 29. Gomes BP, Ferraz CC, Vianna ME, Berber VB, Teixeira FB, Souza-Filho FJ. In vitro antimicrobial activity of several concentrations of sodium hypochlorite and chlorhexidine gluconate in the elimination of Enterococcus faecalis. Int Endod J 2001;34:424–428. 30. Vianna ME, Gomes BP, Berber VB, Zaia AA, Ferraz CC, de Souza-Filho FJ. In vitro evaluation of the antimicrobial activity of chlorhexidine and sodium hypochlorite. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97:79–84. 31. Petri 3rd WH, Wilson TM. Clinical evaluation of antibiotic-supplemented bone allograft. J Oral Maxillofac Surg 1993;51:982–985. 32. Petri 3rd WH, Schaberg SJ. The effects of antibiotic-supplemented bone allografts on contaminated, partially avulsive fractures of the canine ulna. J Oral Maxillofac Surg 1984;42:699–704. 33. White RR, Hays GL, Janer LR. Residual antimicrobial activity after canal irrigation with chlorhexidine. J Endod 1997;23:229–231. 34. Witsø E, Persen L, Løseth K, Benum P, Bergh K. Cancellous bone as an antibiotic carrier. Acta Orthop Scand 2000;71:80–84.
VOLUME 45 • NUMBER 2 • FEBRUARY 2014
