Critical size defects in the long bones of rabbits have been used for years as an experimental model for the investigation of different bone substitute materials. However, no standard surgical protocol exists in the literature.This is a source of misunderstandings and makes results from different studies hardly comparable. This technical note attempts to present a standard surgical technique for the creation of a segmental critical size ulnar defect in the New Zealand white rabbit

Belyaev, Orlin

Huber, Colette

Huber, Franz-Xaver

Meeder, Peter-Juergen

English

Journals from University of Tartu

IntroductionA critical size bony defect (CSD) is defined as thesmallest intra-osseous wound characterized by anabsence of spontaneous healing, which would notheal by bone formation during the lifetime (Schmitz& Hollinger, 1986). In the preclinical field of ortho-pedic and trauma surgery, CSD creation is themethod of choice for in vivo testing of bone repairmaterials (BRM) (Hollinger & Kleinschmidt,1990). Besides in vitro screening methods it isessential to evaluate the biocompatibility of BRMsby in vivo investigations before subjecting them tothe rigors of clinical testing. Only the complexorganism of an animal allows an investigation ofsystemic responses induced by the compleximplant-/transplant-tissue interactions like elimina-tion and bioabsorption kinetics, inflammation reac-tions, tumour genesis and biomechanical effects.Considering this, bone defect models play a crucialrole for experimental testing of orthopaedicimplants, transplants and growth factors. However,a review of the bone research literature reveals anobvious lack of either uniformity or consistencyamong the various researchers concerning animalmodels. CSDs have been determined in variousbones of rats, rabbits, cats, dogs, swine, horses, andnonhuman primate species. Rabbits have beenwidely used in experimental orthopedics and trau-matology, but only a few scientific data exist con-cerning a standard, generally accepted surgicaltechnique of CSD creation in long bones, and inparticular the ulnar leporine bone.Our experience with different surgical techniques ina large number of various experimental animalspecies has led us to develop of a standard, safe,reliable, and repeatable operative protocol for CSDcreation in the ulna of New Zealand white rabbits.The aim of this article is to describe the technicaldetails of this surgical protocol. Materials and methodsGeneral considerationsAll animal experiments were performed in accor-dance with The Council Directive of the EuropeanCommunities on the Protection of Animals Usedfor Experimental and Other Scientific Purposes,1986. Permission was granted from the ethics com-mittee in Karlsruhe (AZ 35-9185.81/54/01). EachScand. J. Lab. Anim. Sci. 2006 Vol. 33 No. 2Published in the Scandinavian Journal of Laboratory Animal Science - an international journal of laboratory animal science89A Standard Surgical Protocol for a Rabbit Ulnar Osteotomy Modelby Franz-Xaver Huber*, Orlin Belyaev, Colette Huber, Peter-Juergen MeederSurgical Clinic, Division of Traumatology and Reconstructive Surgery, University of Heidelberg, Heidelberg, GermanySummary Critical size defects in the long bones of rabbits have been used for years as an experimental model for theinvestigation of different bone substitute materials. However, no standard surgical protocol exists in the lit-erature. This is a source of misunderstandings and makes results from different studies hardly comparable.This technical note attempts to present a standard surgical technique for the creation of a segmental criti-cal size ulnar defect in the New Zealand white rabbit.*Correspondence: Franz-Xaver Huber, M.D.Surgical Clinic, Division of Traumatology andReconstructive Surgery, University of Heidelberg, INF110, 69120 Heidelberg, GermanyTel: +49 6221 566110Fax: +49 6221 564894E-mail: franz-xaver.huber@med.uni-heidelberg.deJD 80 A Standard Surgical  28/06/06  13:58  Side 89Scand. J. Lab. Anim. Sci. 2006 Vol. 33 No. 290study has its own design which is specific for theaims of the experiment: That is why we shall delib-erately limit the subject of this work to a descriptionof the surgical technique and medications directlyrelated to the procedure of the creation of a CSD.MedicationThe operative procedure requires general anesthe-sia. It is induced by atropine (AtropinsulfateBraun®; B Braun, Melsungen, Germany) 0.1mg/kgs.c., xylazine 2% (Rompun®; Bayer, Leverkusen,Germany) 4mg/kg s.c., and ketamine 10%(Ketanest®; WDT, Garbsen, Germany) 60mg/kgi.m.. Together with premedication, the first dose ofantibiotic prophylaxis is given: enrofloxacin 2.5%sol. (Baytril®; Bayer, Leverkusen, Germany)5mg/kg i.m. and the same dose should be repeatedon the first and the second postoperative days.Anesthesia is further maintained by isoflurane-nitrous oxide-oxygen mixture administered bymask. Isoflurane (Forene®; Abbott Rungis, France)2Vol% is used initially for about 5 minutes, then1Vol% for the next 5 minutes and 0.8Vol% for therest of the time. Oxygen is supplied at 800ml/min,and nitrous oxide at 1000ml/min. It is important tonote that pure oxygen should be given alone for thelast three minutes of anesthesia. As anaesthesiologydevice we use the Anesthesia Gas Machine“Narkomat” (Heyer Medical Co., Bad Ems,Germany). Dexpanthenol (Bepanthen® 5g oph-thalmic ointment; Roche, Eppstein-Bremthal,Germany) is placed gently onto the conjunctiva ofeach eye as corneal protection. Carprofen(Rimadyl®; Pfizer, Karlsruhe, Germany) 4mg/kgs.c. once a day is used as analgesic for the first twopostoperative days.Operative procedureThe animal is placed in a true lateral position on thesurgery table on a warming pad, so that the leg to beoperated upon is on top, leaving plenty of room onthe table near the head (Figure 1A). The rabbit’sforeleg is shaved and prepared for surgery by threetimes using a chlorhexidine scrub brush workingfrom the incision site outward. The shaved area isfinally sprayed with betadine (Octanisept), and theincision site is carefully draped to create a sterilearea. A non-sterile assistant holds up the rabbit’s legwhile a sterile surgeon wraps the pad in a small ster-ile drape towel (Figure 1B). With the rabbit’s legstill held up, a sterile drape is placed longitudinallybeneath the leg and a second drape towel is used tocover the rabbit to keep fur out of the sterile area asshown at the picture (Figure 1C), while ensuring thedrapes do not stick to the animal’s whiskers (vibris-sae) or the mask. Finally, a large laparotomy drapeis placed over the rabbit exposing only the rabbit’sleg and ensuring a spacious aseptic working area.To start the operation, the olecranon should be firstpalpated as a point of orientation. Distally the prox-imal part of the shaft of the ulna is palpated and aFigure 1A: Position of the animal on the table.Figure 1B: Covering the operative field.JD 80 A Standard Surgical  28/06/06  13:58  Side 90Scand. J. Lab. Anim. Sci. 2006 Vol. 33 No. 2one inch long, anterolateral slightly curved inci-sion; through skin is made over it with the scalpel.The superficial and deep fasciae are identified andcut separately in line with the skin incision, then a10mm segment of the bone is exposed by dissectionbetween the flexion and extension musculatureoverlying it (Figure 2A). To get a better view of thesitus we use a small, subperiostal Hohmann’s eleva-tors and Langenbeck’s retractor to hold apart mus-cles and soft tissue. The periosteum is incised cir-cumferentially and removed by scraping and clean-ing with a gauze. This is essential for a CSD, sinceperiosteum includes in its cambium layer a highpotential for bone regeneration. Following exposureof the bone a cut of 7 mm length and 5 mm depth ismade using an oscillating bone saw with trephineand trephine guide, thus creating a segmental ulnardefect (Figure 2B, 2C). The trephine is fixed withthe help of a template, taking care to avoid injury tothe radius. The bone marrow is removed with asharp spoon leaving a CSD surrounded by a thinlayer of cortical bone (Figure 2D). Constant copi-ous irrigation with physiological saline solution isused to reduce local heat and protect soft tissuefrom bony debris contamination. A three-hole tita-nium mini-plate is then fixed onto the bone toensure the stability of the defect (Synthes /Oberndorf-Switzerland). Proximally, a 1.1mm hole91Figure 1C: The operative field is already covered.Figure 2A: Access to the ulna.Figure 2B: Performing osteotomy.Figure 2D: CSD – intraoperative situs without afilling.Figure 2C: Schematic presentation of the criticalsize defect in a rabbit’s ulna. JD 80 A Standard Surgical  28/06/06  13:58  Side 91Scand. J. Lab. Anim. Sci. 2006 Vol. 33 No. 292for the screw (1.5mm diameter) is drilled withoutcutting a thread for it. Then the plate is proximallyscrewed, but loose so it can be rotated cranially toallow filling the defect with the studied bone sub-stitute in the form of cement, paste etc (Figure 2E).Then, the free end of the plate is rotated back andadjusted to the distal end of the bone segment,where a second 1.1mm hole is prepared for the sec-ond 1.5mm screw. Both screws are then firmlytightened. The proximal screw should be 6-8mmlong, while the distal one should not exceed 6-7mm(Figure 2F).The multilayer closure of the wound is done withfast absorbable Vicryl Rapide® (Ethicon Inc.) orBiosyn® sutures (Braun-Dexon, Tuttlingen,Germany). The deep and superficial fasciae arecarefully restored with 4-0 single sutures, and 3-0single sutures are used for the skin (Figure 2G). Thearea is sprayed with betadine. An elastic bandage isplaced onto the leg and stuck with plaster. The plas-ter should be stuck not at a slant to avoid compres-sion of the leg (Figure 3). Free movement of the ani-Figure 2E: Stable plate osteosynthesis.Figure 2G: Wound closure – final view.Figure 3: Wound dressing with bandage.Figure 2F: A screw for the osteosynthesis.JD 80 A Standard Surgical  28/06/06  13:58  Side 92Scand. J. Lab. Anim. Sci. 2006 Vol. 33 No. 293mal is allowed immediately after the operation. Thedressing is checked daily for the first 48 hours andthen removed. Conventional radiographs are con-ducted postoperatively. One surgeon should per-form all procedures per experiment to achieve whatis considered to be a uniform technique.Results In a series of 32 consecutive animals we found thatthe above described technique gave excellent intra-and postoperative results. The mean body weight ofthe rabbits was 4.3 (± 0.2) kg. All of them were pre-operatively radiographically screened for bonematurity. The mean operative time was 37.5 (± 4.8)minutes. The clinical progression of the rabbits wasfree of complications. Intraoperative problemsoccurred only in two cases – the point of the screwdrill broke, but was easily extracted from the hole.Other intraoperative complications and problemswere not encountered. There was no postoperativemortality for a period of 60 days. Morbidity waspresented by two cases of delayed wound healingdue to bandage friction. The animals were able tomove freely after the operation. Unproblematicwound healing and bone formation allowed tissuesampling at the end of the study period in 100% ofthe animals. Titanium plates were removed withouttechnical difficulties. DiscussionTesting of BRMs has had an interesting and diversehistory. Bone researchers today cannot agree on thebest model for testing BRMs. Groups studyingceramic compounds have taken a somewhat differ-ent route from those testing osteoinductive proteins,which is different from those testing electrical stim-uli. Frame (1980) provided the classic study on themodel of rabbit’s calvaria when he described thehealing of 5, 10, 15, and 20mm defects in rabbits’calvariae and determined that calvarial defects15mm or greater would not heal by bone formation.Creation of CSDs in the long bones of New Zealandwhite rabbits was first studied by Ben-Fu and Xue-Ming in Shanghai (Ben-fu & Xue-Ming, 1986).They created 6mm defects in the leporine distalradius, which showed no bony growth at 150 daysand thus proved that relatively small osteotomies inthe long bones of rabbits are an excellent model fora CSD. These defects are large enough that doseresponse measurements can be made to optimizethe material for more rigorous testing in later mod-els. Rabbits offer some obvious advantages as a model.They are relatively inexpensive, and plentiful.Compared to rats, which indicate the lower limitappropriate for bone experiments, leporine longbones are big enough to allow surgery without theuse of microinstruments. Furthermore, rats growconstantly throughout their life, which is not thecase with rabbits and humans. Because of the tech-nically demanding surgical procedure many rats arelost before the end of the experiment. Compared tolarger animals, rabbits are easier to handle, lower incosts, and widespread spread experimental modelsin biocompatibility testing. BRMs designed fororthopaedic use can be tested in the long bones ofrabbits, instead of dogs and cats. The rising publicsentiment against the using of “pet” animals such asdogs and cats in laboratory testing may require us tolook at the rabbit in more depth as a bone healingmodel.Fixation of osteotomied bones is an issue thatdeserves special attention. Although the pioneersBen-fu and Xue-Ming, as well as many others afterthem, used no fixation, we consider rigid fixationessential for the accuracy and reproducibility of themethod. It is important when developing a CSDmodel because it is in the first few days of bonyhealing that a critical period exists in which cellsare migrating, attaching, and differentiating intoosteoblasts needed to propagate bone growth.During this time, rigid fixation must be present tooptimize this process and assure maximal boneregeneration (Hollinger & Kleinschmidt, 1990;Reddi et al., 1987). While rigid fixation is neededfor a CSD, each type of fixation has advantages anddisadvantages. The intramedulary fixation devicehas the disadvantage of occluding the bone marrowJD 80 A Standard Surgical  28/06/06  13:58  Side 93Scand. J. Lab. Anim. Sci. 2006 Vol. 33 No. 294and possibly blocking the source of osteoprogenitorcells needed for a BRM to be active. External fixa-tion is undesirable in most animals because theypick and chew on the fixator, resulting in movementof the bone fragments. Infectious complicationswith all devices are possible as well. Our resultsshowed that internal plates and screws function welland are preferable not only in humans, but in ourexperimental leporine model too. We want to pay special attention to the suture tech-nique used for restoring the soft tissues at the end ofthe operation. Our personal experience and com-munications with other authors make us believe thata key to minimizing postoperative morbidity, main-ly wound infections, is the layer-by-layer carefuland atraumatic wound-closure. Pedantic asepsis,meticulous preparation technique and anatomicalrestoration of the dissected structures are crucial insuch models, based on implantation of foreignmaterials.A limitation of the method, characteristic for allanimal bone models, is the varying degree of bonewound repair that is seen along the phylogeneticscale. Any testing scheme would have to take intoconsideration the lesser ability of spontaneous bonywound healing by higher order species (Enneking etal., 1975).Another important detail is the necessity to provethe skeletal maturity of the rabbit before the opera-tion. The animals should be with closed epiphysealplates. Immature animals of most species heal at afaster rate than adults. By testing BRMs in imma-ture animals, falsely high expectations of the mate-rials could be realized. Therefore, it is prudent toassure closure of the epiphysis in all animals radi-ographically prior to their use in an experiment.Weight charts often give a premature indication ofskeletal maturity. In conclusion, we should assess every animal modelform the standpoint of the Three R’s principles –reduce, refine and replace. It should always beemphasized and complied with to ensure the use ofa minimum number of animals, the highest well-being of involved animals, and the use of alternativein-vitro methods (Russell & Burch, 1992). The firstprinciple is achieved in our method by the ability touse the animals further for other experiments, to useulna bilaterally, by avoiding unnecessary duplica-tion of prior research, and by reducing postopera-tive mortality and morbidity, thus decreasing thenumber of animals needed per experiment. The useof rabbits replaces the use of “pet” animals such ascats, dogs, horses and non-human primates whilepreserving the opportunity to perform a study in awhole organism. Our model focuses mainly on therefinement of the experiment procedures, especial-ly the surgical technique used to minimize pain anddistress.Searching for an ideal animal experimental modelin bone research will continue in the future, despitethe significant progress in alternative models and invitro experiments. Testing a bone substitute materi-al in a living organism is a necessary evil beforefurther application in humans. Scientists shouldestablish standard study protocols, and in particularoperative protocols, in order to make results fromthe constantly increasing number of experimentalstudies comparable and thus more useful.JD 80 A Standard Surgical  28/06/06  13:58  Side 94Scand. J. Lab. Anim. Sci. 2006 Vol. 33 No. 295ReferencesBen-fu C & T Xue-Ming: Ultrastructural investiga-tion of experimental non-union of fractures.Chinese Med J, 1986, 99, 207-214.Enneking WF, H Burchardt, JJ Puhl & GPiotrowski: Physical and biological aspects ofrepair in dog cortical-bone transplants. J BoneJoint Surg, 1975, 57A, 237-252.Frame JW: A convenient animal model for testingbone substitute materials. J Oral Surg, 1980, 38,176-180.Hollinger JO & JC Kleinschmidt: The critical sizedefect as an experimental model to test bonerepair materials. J Craniofac Surg, 1990, 1(1), 60-68.Reddi AH, S Wientroub & N Muthukumaran:Biologic principles of bone induction. OrthopClin North Am, 1987, 18, 207-212.Russell W & R Burch: Principles of HumaneExperimental Techniques, UFAW SpecialEdition 1992, Publrs UFAW.Schmitz JP & JO Hollinger: The critical size defectas an experimental model for craniomandibulo-facial nonunions. Clin Orthop, 1986, 205, 299-308.JD 80 A Standard Surgical  28/06/06  13:58  Side 95

A Standard Surgical Protocol for a Rabbit Ulnar Osteotomy Model

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A Standard Surgical Protocol for a Rabbit Ulnar Osteotomy Model

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