Expeimental model for osteotomy in immature rabbit

Abstracts

Apresenta-se um modelo experimental de osteotomia de fíbula em coelhos com esqueleto imaturo adequado para o estudo do processo de reparação óssea. A osteotomia foi realizada por serra elétrica padronizada, sem utilização de dispositivos de fixação. Este modelo foi testado em cinco coelhos e constou da descrição detalhada de técnica operatória, anestesia e técnica radiográfica. Os animais foram submetidos a eutanásia após cinco semanas e tiveram suas fíbulas encaminhadas para avaliação histológica. Comprovou-se que o modelo é eficiente, simples e de fácil execução, tornando-se opção atraente para todos aqueles que desejam realizar estudos de consolidação óssea durante a imaturidade esquelética.

Consolidação óssea; estudo experimental - coelho; osteotomia

ARTIGO ORIGINAL

Expeimental model for osteotomy in immature rabbit

Marcos Almeida Matos^I; Renato Ribeiro Gonçalves^II; Francisco Pereira Araújo^III

^IAssistant Professor - Escola Bahiana de Medicina; Post graduate student at IOT/FMUSP

^IIPost-graduate student at IOT/FMUSP

^IIIResident and Research Trainee at Escola Bahiana de Medicina

SUMMARY

Authors present an experimental model for fibular osteotomy in immature rabbits usefull to study healing bone process. Osteotomy was produced by means of eletric saw without utilization of fixing devices.

This model was applied in five rabbit were sacrified after five weeks and had their fibular send for histological evaluation. Operative technique, anesthesia, roetigenografic technique were detailed discribed.

The model was proved to be efficient, simple and of easy execution, becoming an atractive option for whoever wish to perform studies of bone healing through skeleon development.

Key words: Bone healing; experimental osteotomy; rabbit

INTRODUCTION

Professionals who work with fractures are preferably good physiologists trying to understand the phenomena taking part in bone healing¹³. However, bone repair of fractures is still an open field for research due to its biological complexity.⁽¹²⁾

Several experimental models described in literature are useful for bone healing study. Some restrictions, however, make classical models inadequate for many study objectives. Rat and mouse, for example, have an anatomy and a bone biology that are different from humans¹⁰. Mammals and primates are difficult do handle and have high cost when large groups are considered. Other small sized animals are technically difficult to produce a fracture or osteotomy and require fixation methods that may influence bone healing^1,2,3,4,6,10. It is also difficult to find specific literature on immature skeleton models.

Based on this, it is presented an experimental model of bone healing in immature skeleton rabbits, using open osteotomy of the fibula. Rabbits are largely used as experimental animals, allowing easy handling as well as moderately large groups are affordable^2,6,10. Besides, the fibular anatomy of the pelvian limb of this animal makes possible to evaluate bone healing without interference of fixation devices. We believe that this model can contribute very much for researchers who are interested in the complex biological phenomenon called bone healing.

MATERIAL AND METHODS

Animals (sample)

Five immature skeleton (epiphysial ring still open) albino New Zealand male rabbits (Oryctologus cuniculus) were used. Their weight averaged 729,16g (500 to 1000g).

Environment

The animals were kept in a bioterium in 2320cm² metallic cages. Each cage had one animal feed with standard ration and water.

Anesthesia

HARKNESS and WAGNER (1993)⁵ method was used. Food, but not water was removed for 8 to 12 hours before anesthesia. Each animal was given 0.2 mg/kg of atropine sulphate in order to reduce vagal tonus. Anesthesia consisted in an injection of 25 to 30 mg/kg of ketamine and 5 to 10 mg/kg of diazepan in the proximal muscle region of the pelvian limb. The induction through this technique is estimated to take 4 to 13 minutes, lasting for up to 60 minutes.⁵

Operative technique

The animal was restrained lying back in an adequate operative table. Trichotomy of lateral (fibular) region and antisepsis with degermant soap and 5% iodinated alcohol of all the limb were performed. The area was delimited by sterile drapes. An approximately 5 mm lateral approach to the pelvian limb was performed, over the skin above the fibula. Following division of skin and sub-cutaneous tissue were performed and fibular muscle fascia opened, the muscles retracted and isolated. The fibula was exposed and the periosteum cleared. A shaft osteotomy was then performed in its cranial portion by means of an electric saw with a pattern blade of 12 mm wide and 0.5 mm thick. Polyvicril 5-0 sutures were placed closing the fascia, and Mononylon 5-0 stitches used for closing skin. No patch was used. This procedure was performed in all the 5 rabbits.

Radiological evaluation

Each animal underwent radiological evaluation, just after the procedure and after a 5 weeks period, when a significant bone repair was anticipated. The radiologic technique used was as described by RIBEIRO, AMSTALDEN, and IZALTO (1993)¹¹, being the radiographies taken with a Toshiba XR6010 / 10mas equipment, with an exposure time of 0.3ms, load of 60Kv, distance of 70cm and used Kodak 13x18 films. Kind and place of osteotomy were observed in the first radiography and bone healing phase or healing delay or pseudoarthrosis in the second.

Histological evaluation

After five weeks the animals were pharmacologically sacrificed with the above described anesthesia technique followed by an intravascular injection of 2 ml of potassium chloride. The fibulas were removed and cleared from soft tissues and placed in 10% formalin solution, being then sent to the histopathology laboratory. There, the fibulas were decalcificated with nitric acid 7.5% and included in paraphine. Sagital cuts about 7ì thick were performed and stained with hematoxilin-eosin method for plain optical microscope examination.

RESULTS

No complication was observed in surgical wound and no change in physical health or behavior was noticeable, making us believe that no incapacity or suffering was caused to the animals.

Osteotomy was performed with no difficulty in all the animals. Radiologic evaluation showed that the site of osteotomy was in fibular shaft bone, and the radiological aspect was always very similar, with bone repair callus formation observed in all cases after five weeks.

Histological evaluation of sites of osteotomy demonstrated that in all cases bone healing was in advanced phase. An intraoperative view of the osteotomy can be observed in Figures 1 and 2. Radiological aspect pre and postoperative can be seen in Figures 3 and 4, and the aspect of the consolidated osteotomy in Figure 5. Figure 6 displays a callus in a very advanced healing phase, five weeks after osteotomy.

DISCUSSION

Many experimental models were used to study fracture consolidation process. The problem is that due to anatomical, biological and technique differences, these models not always give adequate parameters for humans.^(10,12) Successful studies in mouse, horse, rat, cat, dog, sheep, chicken, pigeon, primates, rabbits and less common animals were described ¹⁰. Every model of research has advantages and disadvantages, and the researcher should be wise to retrieve from the literature the better fitting to the proposed study method.

Exact understanding of the factors influencing the biological process of bone healing depends on adequately adapted experimental models. Each model fits better a given kind of study. The importance of the development of new experimental models lays on the need to make available for the researchers a wide variety that can fit the specific objectives.

Some already studied models bring difficulties in gathering trustful data when it is decided to compare them to spontaneous bone healing in humans, particularly in pediatric patients. These difficulties come from the need of using fixation devices, excessive mobility of the fracture when not fixated, difficulties in obtaining uniform fractures when manually produced, animal size, and sometimes even because the lack of ability to deal with the chosen animal.^1,4

Most of the studies in bone healing involve mature skeleton animals being the rat chosen in the majority of them^4,6,10. The advantages are their small size and easy handling. Fractures can be easily produced by manual means, however a technical difficulty is usually found in the standardization^4,6. Yet surgical procedures with so small sized animals are difficult and additionally osteotomy or fractures in these animals usually require fixation (external or internal) and this leads to a healing model that is influenced by fixation devices.^(4,10)

Rats and mice have a healing process that is different from humans, since they haven't a Harvesian bone system preventing studies aiming to evaluate the form, function or material and biomechanical properties of Harvesian system¹⁰. It is difficult to get multiple samples of a same bone in these small rodents, that also are not practical for biomechanics and biomaterial tests for requiring difficult methods for fracture or osteotomy fixation^(6,10).

Large animals such as mammals, dogs and primates request expensive costs for their use. It is important that a number large enough of animals is used, in order to reach a statistic significance⁹. Twenty, thirty, or forty large sized animals in a research can be very expensive, and handling them can be very difficult for a regular investigator.

Rabbits however are animals to what most of the researchers are used. Their size is small enough to allow the use of moderately large number of animals, but large enough to allow fixation of the fracture or osteotomy, para-enteral drug administration and biophysical experimentation. Rabbits also have an Harvesian bone system that is similar to human's, so the kind of bone healing after a fracture is very similar to human's one ^{(1,2,5,10,12)}

In this model the rabbit was chosen for being easy to handle, having a size allowing the technical execution of the osteotomy, and because placing them in a cage allows a life closer to the normal. The use of an animal with immature skeleton makes this model peculiar for the study of bone healing when the objective is to extrapolate the data for human pediatric population.

The fractures were surgically executed by means of an electric saw, presenting the same pattern. The traces were similar in their shape and bone location, what is mentioned by some authors as necessary for comparability in performing this kind of study. ^4,8. For this reason, this was not a technical difficulty of this method. The anatomy of the pelvian limb of the rabbit, with a fusion of tibia and fibula in their distal portion allowed to avoid the use a fixation, since the animal could walk normally without noticeable prejudices. The lack of fixation is also desirable in order to observe the natural formation of bone callus without concerns on interference of fixation material^{.3, 4}

It is estimated that in 6 weeks a hole of 5.5 mm produced in a dog tibial metaphysis will have completed its healing proccess¹¹ but the same was not completely observed in rabbits in a similar experimentation by KATTHAGEN & MITELMEIER⁷. Probably, the fact that these authors used a 6 mm hole may explain it, since the hole was larger both physical and relatively ^7,11. So, in this case we decided to use a similar time, however slightly shorter, since these were immature animals with a greater regenerative potential. This time showed to be enough since after five weeks all the animals had a radiographic callus and the histological study demonstrated that healing process was advanced.

Optimization of the model was performed with five rabbits in order to avoid unnecessary losses⁹, since it was objectively uniform in all animals. It is believed that starting from this optimization, larger experiments can be performed for applied studies, such as those to evaluate the effect of drugs and fixation methods over bone healing, particularly in immature skeleton.

CONCLUSION

The experimental model presented for study of bone healing, using fibular osteotomy in immature rabbits is an easy to perform technique that allows the use of a relatively large number of animals at affordable costs. This model will probably contribute to the study of bone healing under drug effects and under physio and pathological different conditions over growing animals.

REFERÊNCIAS

Trabalho recebido em 20/12/2000. Aprovado em 12/06/2001

This work was performed at Escola Bahiana de Medicina and Instituto de Ortopedia e Traumatologia from Faculdade de Medicina da Universidade de São Paulo.

• 1. Bonnarens, F. & Einhorn, T.A.: Production of a standard closed fracture in laboratoryanimal bone. J Orthop Res, 2: 97-101, 1984.
• 2. Brighton, C.T., Horzack, W.J., Brager, M.D., et al: Fracture healing in the rabbit fibula when subjected to various capacitively electrical fields. J Orthop Res, 3:331-340, 1985.
• 3. Camargo, J.N.J.: Estudo experimental comparativo da morfologia e da dinâmica da população celular no calo de reparação de fraturas de ossos longos tratados com e sem osteossíntese intramedular. Tese apresentada à Faculdade de Medicina da Universidade de São Paulo, 1972.
• 4. Guarniero, R.: Estudo da consolidação de fraturas na desnutrição protéica: trabalho experimental em ratos, São Paulo.Tese de Doutorado - Faculdade de Medicina da Universidade de São Paulo, 1987.
• 5. Harkness,J.E. & Wagner,J.E.: Biologia e clínica de coelhos e roedores Ed. Roca, 3şed., São Paulo-SP, 1993.
• 6. Hiltunen, A., Vuorio, E., Aro, H.T.: A standardized experimental fracture in mouse tibia. J Orthop Res, 11:305-312, 1993.
• 7. Katthagen, B. D. & Mitelmeier, H.: Experimental animal investigation of bone regneration with collagen-apatite. Arch Orthop Trauma Surg, 103:291-302,1984.
• 8. Köberle,G.: Estudos físicos e biológicos em tecido ósseo. Tese apresentada à Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, 1974.
• 9. Leopizzi, N, Bollinger Neto, R., Barros Filho, T.E.P., Azze, R.J.: Modelos experimentais em ortopedia e ISO 10993: BiologicaL evaluation of medical devices part 2: animal walfare requirements. Interpretação e extensão da norma. Acta Ortop Bras 6:139-142, 1998.
• 10. Nunamaker, V.M.D.: Experimental model of fracture repair. Clin Orthop, 355S:57-65, 1998.
• 11. Ribeiro, R.F.; Amstalden, E.M.I.& Izalto, I.C.: Modelo experimental de regeneração óssea espontânea. Rev Bras Ortop, 31: 931-935, 1996.
• 12. Turek,S.L.: Reparo da fratura simples de um osso longo. in Ortopedi - Princípios e sua aplicação . São Paulo, Manole ltda, 1991. Cap.3, p.58-62.
• 13. Watson-Jones, R.: Fractures and joint injuries. 5^th ed. London, Churchill Livingstone,1976.

Publication Dates

• Publication in this collection
  13 Oct 2005
• Date of issue
  Dec 2001

History

• Accepted
  12 June 2001
• Received
  20 Dec 2000