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An animal model in sheep for biocompatibility testing of biomaterials in cancellous bones
© Nuss et al; licensee BioMed Central Ltd. 2006
Received: 27 March 2006
Accepted: 15 August 2006
Published: 15 August 2006
The past years have seen the development of many synthetic bone replacements. To test their biocompatibility and ability for osseointegration, osseoinduction and -conduction requires their placement within bone preferably in an animal experiment of a higher species.
A suitable experimental animal model in sheep with drill holes of 8 mm diameter and 13 mm depth within the proximal and distal humerus and femur for testing biocompatibility issues is introduced.
This present sheep model allows the placing of up to 8 different test materials within one animal and because of the standardization of the bone defect, routine evaluation by means of histomorphometry is easily conducted. This method was used successfully in 66 White Alpine Sheep. When the drill holes were correctly placed no complications such as spontaneous fractures were encountered.
This experimental animal model serves an excellent basis for testing the biocompatibility of novel biomaterials to be used as bone replacement or new bone formation enhancing materials.
The use of resorbable and non- resorbable biomaterials, such as hydroxyapatite or tricalcium phosphate, as synthetic bone replacements is well established in orthopaedic, maxillofacial and dental surgery [1, 2]. Although autologous cancellous bone is the material of choice for bone replacement and induction there are limitations in relation to its use, such as limited amount of material, additional surgical procedure, prolonged surgery and complications of wound healing at the donor site. Because of this there is continuous interest in the development of new synthetic materials [3–5].
The most important aspect in the development of new (biodegradable) biomaterials is the experimental and clinical testing for biocompatibility [6–8]. This is closely followed by the bioactivity, which relates to the resorption or integration of the implanted material into the surrounding bone, also called "osseointegration" and the ability to initiate and support the apposition and integration of the new bone, in particular in comparison with previously established materials .
Various animal species are used for these biocompatibility tests, such as the mouse , rat [10–18], guinea pig , rabbit [20–23], dog [24–28] goat  and sheep [30–32]. Furthermore, different implantation sites and methods have been used to examine biocompatibility issues. Among them are intra-peritoneal , subcutaneous [12, 14, 17, 33], intraosseous such as in the mandible [19, 27], femur [11, 15, 24, 25]), tibia [18, 20, 21, 23, 26, 30], cranial bone [28, 31] and intramuscular [9, 16, 29] applications.
The use of sheep for orthopaedic research continues to increase. This is due to the similarities with humans in weight, bone and joint structure and bone regeneration [34–36]. Although rodents may be less expensive, they have different bone morphology. Another practical reason is that rodents often are too small in size to test degradable materials in bone especially in combination with internal fixation and fracture repair [37, 38]. Furthermore, the influence of the different mechanical properties of bone from different species awaits clarification in relation to the outcome of these studies [39, 40].
To contribute to the standardisation of testing new and biodegradable materials for use in orthopaedics, maxillofacial and dental surgery our research group has developed an animal model with sheep that allows the intraosseous implantation of 8 different samples per sheep in long bones [41, 42]. This animal model facilitates testing inter- and intra-individual differences among different materials while at the same time reducing overall suffering of animals as well as necessary numbers to satisfy statistical requirements. It has been already successfully applied for several studies related to testing biodegradable materials  and to the knowledge of the authors has never been described before in the literature.
Implanted were various cements (β-tricalciumphosphates, brushite, hydroxyapatite), hydrogels (fibrin-based, polyethylene glycol) and other resorbable or non-resorbable bone replacement materials as composites with or without growth factors such as parathyroid hormone (PTH1–34), bone morphogenic protein (BMP-2), transforming growth factor (TGFβ) and insulin-like growth factor 1 (IGF-1).
The experimental animals were kept in stalls prior to surgery. Food was withdrawn 36 hours, and water 6 hours prior to anaesthesia. A pre-anaesthetic examination was performed including haematology and a chemscreen consisting of liver enzymes, urea, creatinine, protein, albumin, sodium and potassium. After recovery of the animals from anaesthesia, they were initially kept in small groups in stables. After 10 days, the skin staples were removed and the animals were allowed to join the flock on the fields normally at 20 days after surgery. They were checked daily for lameness and additional clinical problems. Food and water were given ad libitum.
Animals were sedated with medetomidine (5 μ g/kg BW, Domitor™, Orion Pharma Animal Health, Finland) and anaesthesia was induced with ketamine (2 mg/kg BW, Narketan® 10, Chassot GmbH, Germany) in combination with diazepam (0.01 mg/kg BW, Valium®, Roche, Switzerland) and maintained with 0.8 Vol% isoflurane (Forene®, Abbot AG, Switzerland) in O2 and an infusion of Ringer's solution with 60 mg ketamine (Narketan™ 10, Chassot GmbH, Germany)/litre at a rate of 10 ml/kg BW/hour. The animals received as pre-and post-operative prophylaxis 30,000 IU penicillin/kg BW (Hoechst AG, Germany) and 6 mg gentamicin/kg BW (Streuli & Co AG, Switzerland) intravenously twice a day. In addition, they received subcutaneously 500 Units of equine tetanus serum as a single application (Tetanus Serum Veterinaria AG, Zurich, Switzerland).
Analgesia was maintained through injection of 0.01 mg buprenorphine/kg BW i.v. perioperatively and additionally 4 mg carprofen/kg BW i.v. (Rimadyl®, Pfizer Inc., NY, USA) postoperatively for 3 days. The area in the region of the incisions was clipped and disinfected in the standard manner.
The animals were placed in left or right lateral recumbency on the operating table with the limbs placed in a horizontal position. The approach to the bone was always from the lateral side (Fig. 2). It is important for the forelimb that the humerus is positioned parallel with the humeral condyles being at a 90° angle to the surgery table. Furthermore the limb should be slightly flexed to the position that the lateral epicondyle of the humeral condyle can be palpated easily directly under the skin. The hind limb should be also slightly flexed such that the lateral collateral ligament can be palpated through the skin, although a slight downward inclination of the hind limb may facilitate access to the proximal femur. Both, the fore- and hind limbs should be additionally supported from underneath so that they cannot be pushed down when drilling the hole but remain stable in the prepared plane.
With all approaches an attempt was made to keep the skin incisions and preparation of soft tissue down to the bone as small as possible (ca. 6–8 cm in length). To facilitate the further surgical approaches to all locations, the wounds were kept open by means of either a Weitlaner retractor alone or in combination with a Gelpi retractor that was usually placed perpendicular to the already placed Weitlaner retractor (proximal and distal femur). The periosteal elevator was used to prepare the location of the drill hole such that all overlying soft tissues were removed from the bone. This facilitated the placement of the drill guide and avoided the slippage of the drill bit initially before the drill had started to penetrate the cortical bone. The description of the four different surgical sites is given below. A total number of 560 drill holes were placed (n = 560 with 70 drill holes in each location). Immediately after drilling, the holes were filled with the various test materials. After filling the drill holes, the different wound layers as described in the approaches were closed separately with non-resorbable suture material (Vicryl 2/0, Johnson & Johnson, Brussels, Belgium) and the skin was stapled (Davis and Geck Appose ULCr, B. Braun Aesculap AG, Tuttlingen, Germany)
Proximal part of humeral diaphysis (Fig. 3)
Distal epiphysis of the humerus (Fig. 4)
Proximal part of the femoral diaphysis (Fig. 5)
Distal epiphysis of the femur (Fig. 6)
Evaluation of samples
Anaesthesia and recovery phases were uneventful for 51 of the 55 animals. As soon as the sheep were awake they were placed in straw-lined small stalls. Ten days later, after removal of the skin staples, animals were released to larger stalls and finally were allowed to roam free on pasture after 20 days. At no time did they show signs of lameness or other discomfort.
For 4 sheep, the positioning of the drill holes was not exactly in the correct places described above, but slightly off towards the diaphysis of the femur or distal humerus respectively. These animals suffered either from a comminuted fractured humerus (n = 1) or femur (n = 3) within the first 3 postoperative days. All four animals were immediately euthanized after the complications occurred. All other drill holes (total of n = 528 holes) could be easily placed except in the distal humerus, where in 9 instances the drill hole was initiated too far cranially and distally resulting in slippage of the drill bit into the cranial pouch of the elbow joint including an incomplete drill hole within the cranial aspect of the lateral humeral condyle. In those 9 instances, where initial slippage of the drill bit occurred from the lateral humeral condylus, the surgeon gave up drilling the hole to avoid spontaneous fractures of the distal humerus. There, the biomaterials were never applied.
After the pre-planned time period the animals were slaughtered and the treated bones removed. Depending on the time point of sacrifice the original drill hole was easily detected or especially at later time points almost healed, at least at the cortical site. But even then, there was a small amount of connective tissue visible in the area indicating the original defect within the cortical bone. No macroscopic signs of inflammatory reactions were generally seen even at the early time points. However, this was dependent on the implanted material . Normally, the radiographs taken with the faxitron machine revealed the location and direction of the drill hole, although at later time points (24 weeks) depending on the biomaterials bone healing could be so far advanced that detection proved to be difficult and only signs of intensive bone remodelling indicated the original bone defects.
The time frame up to 6 months proved to be adequate for this size of defect, since most of the biomaterials tested were resorbed and replaced by new bone to at least two thirds of the original bone defect within this period. The same was true for the assessment of cellular events within this given time point. Degradation mechanisms could be easily followed at the interface between biomaterials and host tissue. The early time points (2, 4 weeks) were helpful to test biocompatibility issues since the appearance of foreign body or other mononuclear cells indicative of host reaction to the material occurred mainly within this early phase.
The drill hole model in sheep as presented is well suited to test biocompatibility issues regarding biodegradable materials, but also to answer questions related to material resorption, substitution with new bone or less functional tissue such as fibrous tissue. The experimental model was established in 70 sheep with a total of 560 drill holes for materials to be tested in bone. Several advantages are pertinent to this well standardized animal model with animal welfare being one of the most important aspects, closely followed by the possibility to translate results relatively easy to humans without the necessity of additional animal experiments related to biocompatibility questions.
The surgical technique requires a modified drill bit with a flattened tip, solid anatomical knowledge and experience of the surgeon. The small stab incisions and minimal preparation of soft tissues to access the bone cortex, where the drill hole will be placed, are part of the regimen of minimally invasive surgery and thus, atraumatic technique including prevention of unnecessary suffering of the animals.
It is common belief that for ethical reasons small experimental animals, such as mice, rats or rabbits, should be used wherever possible. Another reason to turn to small rodents is for economical aspects as rats and mice are much less expensive and easier to maintain as sheep. Biocompatibility of novel materials is often tested in less demanding animal models in the literature. Biological materials implanted into soft tissue, for example intramuscularly [9, 16, 29] or subcutaneously [12, 14, 17, 33]), may answer questions pertaining to the local cellular and humoral reaction, but not to the degree of new bone formation, osteoinduction and -conduction. Even the local cellular degradation mechanisms may be different in soft and osseous tissue, since osteoclasts often involved in synthetic hydroxyapatite-based bone materials, are only present in bone but not subcutaneous or muscular tissues. In addition, any material placed in soft tissue may elicit the formation of a soft tissue capsule in an attempt to wall it off. This reaction most likely has nothing in common with a response related to biocompatibility, but could also be a reaction to mechanical instability and different stiffness and rigidity of soft tissue and the implanted biomaterial. In the author's judgement the subcutaneous and intramuscular pouch model in rodents as introduced by Urist et al [47, 48] is a valuable animal model for heterotopic osteoinduction triggered by biomimetic substances, but not for testing biocompatibility issues related to bone.
There are successful reports with drill holes used in rabbit tibias [23, 26] and rat femurs . Rodents may require less time and cost as well as less sophisticated surgery and anaesthesia facilities. However, more animals per study may be required due to limited anatomical space and size of animals. Furthermore, bone metabolism is significantly different with mostly faster bone remodelling. Positive results in rodents may have to be repeated and verified in larger species before human clinical trials can be initiated. Because of the similarities with human bone metabolism the results from sheep carry more authority than those obtained small laboratory animals .
The drill hole model as presented here has additional advantages. With this model it is possible to investigate and compare the in vivo characteristics of up to eight different materials in one animal. Also it is possible to compare more than one sample of the same material within an animal but in different locations representing different densities of cancellous bone. This is not just a question of animal protection, but also adds the possibility to compare directly the individual's reaction to various materials. Last but not least, this model allows testing of biomaterials without other than the physiologic mechanical load, which is in contrast to animal models that produced single injection or implantation sites using osteotomy or ostectomies [24, 30, 49]. These models may not be as suitable for biocompatibility testing of materials in bone as the drill hole model, since it may be difficult to attribute failures to material incompatibility or mechanical problems. Critical sized bone defects in long or cranial bones are the next step once the biocompatibility of the material has been established [31, 32, 50, 51].
The drill hole model in sheep proves to be an excellent animal model to test biocompatibility of biomaterials that should be implanted in bone. Overall, it is a safe model for the experienced surgeon, reduces suffering and numbers of experimental animals and serves as an excellent basis for testing the best materials in a more challenging animal model where mechanical properties play a role such as in critical sized bone defects of long, maxillofacial or cranial bone.
The authors wish to thank Matthias Haab for the excellent drawings, Katalin Zlinszky and Sabina Wunderlin for establishing the histological techniques, the doctorate students D. Apelt, L. Meinel, F. Theiss, M. Kemper, A. Oberle and O. Genot for support with the animal experiments including establishing the surgical technique and evaluation methods, Adrian Fairburn for his support in writing the English manuscripts, and the sheep for serving as experimental animals.
The studies included in this publication were financed partly (grant for K.N.) by The National Centre of Competence (NCCR) CO-ME, Zurich, Switzerland.
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