Development of an experimental model of infected bone void in the ulna of rabbits

Abstracts

OBJETIVO: Desenvolver um modelo experimental que permita estudar a regeneração de grandes falhas ósseas em condições de infecção. MÉTODO: Falhas ósseas segmentares de 15mm foram criadas cirurgicamente na ulna de 12 coelhos e inoculadas com 5x10(8) unidades formadoras de colônia (UFC) de S. aureus. O desbridamento da infecção foi realizado duas semanas após, seguida da aplicação sistêmica de gentamicina por quatro semanas. Os animais foram acompanhados por um período de 12 semanas para avaliação do controle da infecção e da regeneração óssea. RESULTADOS: A regeneração espontânea foi inferior a 25% do defeito na avaliação radiográfica e histológica. CONCLUSÃO: A Falha óssea infectada de 15mm na ulna de coelhos é incapaz de alcançar a regeneração completa sem tratamentos adicionais. Nível de Evidência V, Estudo experimental.

Doenças ósseas infecciosas; Regeneração óssea; Coelho

ORIGINAL ARTICLE

^IDepartment of Biomechanics, Medicine and Rehabilitation of the Musculoskeletal System, Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo

^IIDepartment of Clinical Medicine, Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo

^IIIDepartment of Surgery, Oral and Maxillofacial Traumatology and Periodontics, Faculdade de Odontologia de Ribeirão Preto da Universidade de São Paulo

Mailing address

ABSTRACT

OBJECTIVE: Develop a model that allowed the study of bone regeneration in infection conditions.

METHOD: A 15 mm defect was surgically created in the rabbit ulna and inoculated with 5x10⁸ colony-forming units (CFU) of S. aureus. Surgical debridement was performed two weeks after and systemic gentamicin was administered for four weeks. Animals were followed up to 12 weeks to evaluate infection control and bone regeneration.

RESULT: Bone regeneration was inferior to 25% of the defect in radiological and histological analysis.

CONCLUSION: Infected bone defect of 15 mm in the rabbit ulna was unable to achieve full regeneration without further treatment. Level of Evidence V, Experimental Study.

Keywords: Bone diseases. Infectious. Bone regeneration. Rabbits.

INTRODUCTION

The loss of large bone segments remains one of the most challenging problems in orthopedic surgery,^1,2 and is frequently associated with infection.^2-5 Experimental models are useful for assessing the efficacy of new treatments, and should be reproducible, well controlled and afford the application of standardized methods of analysis.⁶

Evans, Nelson and Harrison⁷ described a method for creating experimental infection in rabbit forearms, which has become one of the most important for studying osteomyelitis. However, in this model infection produces alterations in the bone architecture such as osteolysis, sequestration, sclerosis and diaphyseal enlargement,^7,8 which render it unsuitable for analyzing regeneration. The aim of this study was to develop a model of infected bone gap/defect that would allow the adequate evaluation of bone regeneration.

MATERIAL AND METHODS

Staphylococcus aureus inoculum

Samples of the selected strain were seeded in blood agar and left in the incubator at a temperature of 35ºC for 24 hours. After this period the material was diluted in sterile saline until it reached the turbidity corresponding to 10¹⁰ CFU/ml. A sample of the solution was then diluted to 10⁴ and read in the Neubauer chamber (Herka Intercolor), confirming the concentration, and seeded in CLED agar to test the viability of the inoculum.

Experimental model

The study was approved by the ethics committee in animal experimentation of Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, and twelve female rabbits weighing between 2.5 and 3.5kg were used. The animals were anesthetized using ketamine hydrochloride (40mg/Kg) and xylazine (8mg/Kg) applied via intramuscular route, followed by continuous venous infusion of 1% ketamine. The right ulna was exposed surgically and a 15mm bone segment from the distal third was resected without the periosteum using a bone saw.

After this the segment was placed back on the defect and 0.05 cc of the inoculum (5x10⁸ CFU) was introduced into the medullary canal using a 0.3 cc syringe. The periosteum and skin were sutured closed and analgesia with ketoprofen (2/mg/Kg IM) was performed for three days after each surgical procedure.

Two weeks later the wound was debrided, the bone fragment collected for culturing, all the necrotic tissues removed and the wound cleaned with 40ml of 0.9% sterile saline solution. Two animals were euthanized soon after the debridement, and the others received 6mg/Kg/day IM gentamicin for four weeks. The bone fragment collected in debridement was cultivated on blood agar and tested for antimicrobial sensitivity (Walkaway-96, Baxter Diagnostics Inc.).

Administration of fluorochromes was performed in two animals, alizarin (10mg/Kg) four weeks after debridement, calcein (10mg/Kg) at eight weeks and oxytetracycline (10mg/Kg) 48 hours before euthanasia.

Radiographs were taken after debridement and at 4 and 8 weeks. The animals were euthanized after 12 weeks with sodium thiopental (50mg/Kg IV) and the piece was submitted to radiography with high resolution film (Kodak Insight^®).

Radiographic and histological analysis

The infection was evaluated radiographically according to the degree of destruction of the bone architecture and of the periosteum⁹ and regeneration based on the size of the bone defect and on the void filling percentage.

For histology the region of the bone void was decalcified with 7.5% nitric acid, soaked in paraffin and sectioned lengthwise. Hematoxylin-eosin and Gram were used for staining.^10,11 The samples with fluorochromes were submitted to dehydration in increasing gradations of alcohol and then were embedded in acrylic resin. The cross-sections were stained with a mixture of methylene blue, azure II and pararosaniline or left unstained for fluorescence.¹² The histological analysis used the scale described by Ambrose et al.¹¹

Statistics

SPSS 15.0 software was used for data analysis with significance level of p = 0.05.

RESULTS

Eleven wounds (92%) had clinical signs of infection at the time of debridement. The average size of the bone defect measured in the intraoperative period, increased from 15mm before the inoculum to 16 mm after debridement (p = 0.01). Two animals died during the follow-up of causes not related to the study. After 12 weeks all the wounds were healed and without signs of infection. S. aureus was the only agent isolated in the samples analyzed. Periosteal reaction was observed on the borders of the bone void in 58% of the animals in the initial radiographs, considered secondary to surgical handling and not to infection. No radiographic alterations related to infection were observed at the end of study. The average length of the bone defect decreased from 16mm after debridement to 11mm at the end of the follow-up. Union (defined as bone regeneration above 25% of the defect) occurred in only one fourth of the bone voids. The mean percentage of gap filling rose from 3% in the fourth week after debridement to 6% after 12 weeks. (Figure 1) The presence of bacteria was not observed in the histological control, yet signs of intraosseus inflammation were identified according to Table 1. After 12 weeks the percentage of void filling was below 25%, with most of the regenerate material originating from the periosteum of the intraosseus membrane. The fluorescence showed that bone deposition occurred predominantly during the first four weeks, and was minimal at 12 weeks. (Figure 2)

DISCUSSION

Rabbit ulna segmental bone defect is a well-established model in the study of bone regeneration.^13-19 Inocula ranging between 10⁵-10⁸ in a segment of devitalized bone produce infection rates above 90%, with the time to debridement ranging from two to four weeks.^7,8,11 Longer periods are associated with intense destruction of the bone architecture and disorganized periosteal reaction, with the infection extending through the bone,^7,8,11,20,21 hindering the radiographic and histological evaluation of regeneration.

In this study infection was obtained in 100% of the animals without important destruction of the bone architecture after two weeks. With debridement there was an increase in the bone void and four weeks of systemic antibiotics were sufficient to control the infection, with no signs of recurrence during follow-up. After 12 weeks the bone regeneration of the void was below 25% both in the radiologic and histological evaluation.

CONCLUSION

An infected bone void of 15 millimeters in rabbit ulna can be considered a "critical void" since spontaneous regeneration is not achieved. This model proved to be predictable and well controlled, and as such, appropriate for studying bone regeneration under infection conditions.

REFERENCES

• 1. Masquelet AC. Muscle reconstruction in reconstructive surgery: soft tissue repair and long bone reconstruction. Langenbecks Arch Surg. 2003;388(5):344-6.
• 2. DeCoster TA, Gehlert RJ, Mikola EA, Pirela-Cruz MA. Management of posttraumatic segmental bone defects. J Am Acad Orthop Surg. 2004;12(1):28-38.
• 3. Gustilo RB, Mendoza RM, Williams DN. Problems in the management of type III (severe) open fractures: a new classification of type III open fractures. J Trauma. 1984;24(8):742-6.
• 4. Goldstrohm GL, Mears DC, Swartz WM. The results of 39 fractures complicated by major segmental bone loss and/or leg length discrepancy. J Trauma. 1984;24(1):50-8.
• 5. Ostermann PA, Henry SL, Seligson D. The role of local antibiotic therapy in the management of compound fractures. Clin Orthop Relat Res. 1993;(295):102-11.
• 6. Lane JM, Sandhu HS. Current approaches to experimental bone grafting. Orthop Clin North Am. 1987;18(2):213-25.
• 7. Evans RP, Nelson CL, Harrison BH. The effect of wound environment on the incidence of acute osteomyelitis. Clin Orthop Relat Res. 1993;(286):289-97.
• 8. Smeltzer MS, Thomas JR, Hickmon SG, Skinner RA, Nelson CL, Griffith D et al. Characterization of a rabbit model of staphylococcal osteomyelitis. J Orthop Res. 1997;15(3):414-21.
• 9. Calhoun JH, Mader JT. Treatment of osteomyelitis with a biodegradable antibiotic implant. Clin Orthop Relat Res. 1997;(341):206-14.
• 10. Skinner RA, Hickmon SG, Nelson CL, Germer RA. Modified stain for identification of Staphylococcus aureus in osteomyelitis. J Histotech. 1992;15:303-6.
• 11. Ambrose CG, Clyburn TA, Louden K, Joseph J, Wright J, Gulati P et al. Effective treatment of osteomyelitis with biodegradable microspheres in a rabbit model. Clin Orthop Relat Res. 2004;(421):293-9
• 12. Sverzut CE, Faria PE, Magdalena CM, Trivellato AE, Mello-Filho FV, Paccola CA et al. Reconstruction of mandibular segmental defects using the guided-bone regeneration technique with polylactide membranes and/or autogenous bone graft: a preliminary study on the influence of membrane permeability. J Oral Maxillofac Surg. 2008;66(4):647-56
• 13. Cook SD, Baffes GC, Wolfe MW, Sampath TK, Rueger DC, Whitecloud TS 3rd. The effect of recombinant human osteogenic protein-1 on healing of large segmental bone defects. J Bone Joint Surg Am. 1994;76(6):827-38.
• 14. Bostrom M, Lane JM, Tomin E, Browne M, Berberian W, Turek T et al. Use of bone morphogenetic protein-2 in the rabbit ulnar nonunion model. Clin Orthop Relat Res. 1996;(327):272-82.
• 15. Perka C, Schultz O, Spitzer RS, Lindenhayn K, Burmester GR, Sittinger M. Segmental bone repair by tissue-engineered periosteal cell transplants with bioresorbable fleece and fibrin scaffolds in rabbits. Biomaterials. 2000;21(11):1145-53.
• 16. Djapic T, Kusec V, Jelic M, Vukicevic S, Pecina M. Compressed homologous cancellous bone and bone morphogenetic protein (BMP)-7 or bone marrow accelerate healing of long-bone critical defects. Int Orthop. 2003;27(6):326-30.
• 17. Kokubo S, Fujimoto R, Yokota S, Fukushima S, Nozaki K, Takahashi K et al. Bone regeneration by recombinant human bone morphogenetic protein-2 and a novel biodegradable carrier in a rabbit ulnar defect model. Biomaterials. 2003;24(9):1643-51.
• 18. Sheller MR, Crowther RS, Kinney JH, Yang J, Di Jorio S, Breunig T et al. Repair of rabbit segmental defects with the thrombin peptide, TP508. J Orthop Res. 2004;22(5):1094-9.
• 19. Seeherman HJ, Azari K, Bidic S, Rogers L, Li XJ, Hollinger JO et al. rhBMP-2 delivered in a calcium phosphate cement accelerates bridging of critical-sized defects in rabbit radii. J Bone Joint Surg Am. 2006;88(7):1553-65.
• 20. Nelson CL, Hickmon SG, Skinner RA. Treatment of experimental osteomyelitis by surgical debridement and the implantation of bioerodable, polyanhydride-gentamicin beads. J Orthop Res. 1997;15(2):249-55.
• 21. Joosten U, Joist A, Frebel T, Brandt B, Diederichs S, von Eiff C. Evaluation of an in situ setting injectable calcium phosphate as a new carrier material for gentamicin in the treatment of chronic osteomyelitis: studies in vitro and in vivo. Biomaterials. 2004;25(18):4287-95.

Development of an experimental model of infected bone void in the ulna of rabbits

Matheus Lemos Azi^I; Mauricio Kfuri Junior^I; Roberto Martinez^II; Luis Antonio Salata^III; Cleber Antonio Jansen PaccolaI,^*

Publication Dates

• Publication in this collection
  02 July 2012
• Date of issue
  2012

History

• Received
  29 May 2010
• Accepted
  27 Apr 2011