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Establishment of a preclinical ovine screening model for the investigation of bone tissue engineering strategies in cancellous and cortical bone defects
© Pobloth et al. 2016
Received: 6 November 2015
Accepted: 24 February 2016
Published: 1 March 2016
New tissue engineering strategies for bone regeneration need to be investigated in a relevant preclinical large animal model before making the translation into human patients. Therefore, our interdisciplinary group established a simplified large animal screening model for intramembranous bone defect regeneration in cancellous and cortical bone.
Related to a well-established model of cancellous drill hole defect regeneration in sheep, both the proximal and distal epimetaphyseal regions of the femur and the humerus were used bilaterally for eight drill hole cancellous defects (Ø 6 mm, 15 mm depth). Several improvements of the surgical procedure and equipment for an easier harvest of samples were invented. For the inclusion of cortical defect regeneration, a total of eight unicortical diaphyseal drill holes (6 mm Ø) were placed in the proximal-lateral and distal-medial parts of the metacarpal (MC) and metatarsal (MT) diaphyseal bone bilaterally. Acting moments within a normal gait cycle in the musculoskeletal lower limb model were compared with the results of the biomechanical in vitro torsion test until failure to ensure a low accidental fracture risk of utilized bones (ANOVA, p < 0.05). The model was tested in vivo, using thirteen adult, female, black-face sheep (Ø 66 kg; ± 5 kg; age ≥ 2.5 years). In a two-step surgical procedure 16 drill holes were performed for the investigation of two different time points within one animal. Defects were left empty, augmented with autologous cancellous bone or soft bone graft substitutes.
The in vitro tests confirmed this model a high comparability between drilled MC and MT bones and a high safety margin until fracture. The exclusion of one animal from the in vivo study, due to a spiral fracture of the left MC bone led to a tolerable failure rate of 8 %.
As a screening tool, promising biomaterials can be tested in this cancellous and cortical bone defect model prior to the application in a more complex treatment site.
KeywordsLarge animal model Drill hole model Cortical and cancellous bone defect regeneration
Although bone possesses a good healing capacity, complications during bone defect regeneration are still a significant clinical concern. Various aspects such as defect size, defect localization, biomechanical conditions, infections, and different underlying diseases affect the healing situation [1, 2]. To promote endogenous bone defect regeneration several strategies are under investigation, e.g. the use of growth factors or scaffold materials for bone tissue engineering [3–5]. A general understanding of these novel therapeutical strategies can be achieved in in vitro cell cultures and in small animal models, but these models are somewhat limited. Feasibility of upsizing promising scaffold solutions to a relevant defect size and adaptation to certain bone structures are reasons for the need of a large animal model before a translation into human patients can be considered. Bone defect regenerative mechanisms, such as direct intramembranous ossification or endochondral ossification, are dependent on the involved bone structure and bone defect size . While larger, diaphyseal bone fractures heal via endochondral bone regeneration, small diaphyseal defects and epimetaphyseal defects affecting the cancellous bone regenerate by direct intramembraneous ossification [7–11]. Additionally, diaphyseal bone essentially heals through periosteal callus formation, while epimetaphyseal bone usually heals endosteally with micro-callus and only minimal periosteal callus formation [8, 9, 12, 13]. New tissue engineering strategies should support the different healing mechanisms involved under consideration of these different requirements. Therefore, a suitable animal model needs to be carefully considered. Several small and large animal models have been established using different defect sizes, critical and non-critical, affecting either the epimetaphyseal or diaphyseal part of long bones [2–5, 14]. However, a validated large animal model offering the possibility to test bone tissue engineering strategies in both cancellous and cortical bone with an adequate sample size is lacking. In this article we show the approach of our multidisciplinary research team to establish a simplified large animal screening model, which allows the analysis of promising biomaterials in both epimetaphyseal and diaphyseal bone simultaneously by using defects of adequate dimension, and at different healing time points, resulting in a reduction of the number of experimental animals.
A drill hole of 6 mm diameter was performed to produce a bone defect size below the recommended 50 % of the corresponding bone diameter (43 – 50 % metacarpal bone; 35–40 % metatarsal bone) for a low fracture risk and an adequate bone defect size for thorough investigations of bone forming biomaterials .
A biomechanical validation of the metacarpal and metatarsal bones used in this model was performed by a torsion test until failure of native bones as well as drilled bones. The native metacarpal and metatarsal bones were compared in this test for a general characterization of the utilized bones. The 6 mm defect size was investigated in the metacarpal and metatarsal bones to confirm that these two drill holes per bone did not result in a stress-riser and bone failure under physiological strains. The drill holes were positioned in the same location as during surgery. Four paired metacarpal and metatarsal bones harvested from four earlier sacrificed adult sheep were used. Eight native bones and the corresponding contralateral bones were each prepared with 6 mm diameter proximal-lateral and distal-medial unicortical drill holes. In order to stabilize the later bone-embedding material compound short k-wires were driven perpendicular through the bone ends. All bones were aligned vertically using a cross-line laser. Afterwards, two centimeters of the bone ends were embedded in the two component plastic Memecryl® (Bauer Handels GmbH, Fehraltdorf, Switzerland) in custom-made test pots. A load to failure test was performed in the Zwick testing machine (Zwick Roell AG, Z010, Ulm, Germany). The test protocol involved an axial pre-load of 0.5 N followed by an axial rotation at a rate of 10 ° / min until failure of the tested structure occurred. Applied rotation and resulting torque were acquired at 10 Hz with the software testExpert® II (Zwick Roell AG, Z010, Ulm, Germany). Subsequent data analysis in Matlab 7.7 (The MathWorks GmbH, Ismaning, Germany) resulted in the maximum torsional moment until failure (Mmax [Nm]), the angle at Mmax [°], the torsional stiffness [Nm/°] as the linear part of the load/deformation curve, and the energy to failure [Nm rad].
Musculoskeletal lower limb model
The previously published musculoskeletal model of the lower limb in sheep was used to calculate the acting torsional moment along the metacarpal bone axis within a complete gait cycle . The moment (Mz) was calculated as 0.0027 body weight per meter (BWm). Expecting the body weight of a black-face sheep to be approximately 85 kg a maximum torsional moment of 2.25 Nm would act on the metacarpal bone during a regular gait cycle. Assuming a safety factor of three, the metacarpal and metatarsal bones would have to withstand a torsional moment of 6.75 Nm during a normal gait cycle.
Thirteen adult, female, black-face sheep (mean weight 66 kg; standard deviation ± 5 kg; age ≥ 2.5 years) were included in the study. All animals received 16 drill holes in total. With a diameter of 6 mm and a depth of 15 mm eight drill holes were performed in the proximal and distal epimetaphyseal bone of the femur and humerus bilaterally. Additionally two diaphyseal, unicortical drill holes (6 mm Ø) were placed in the proximal-lateral and distal-medial diaphysis of the metacarpus (MC) and metatarsus (MT) bilaterally. The drill hole defects in the right humerus, femur, metacarpus and metatarsus were placed in one surgery and defect healing was evaluated nine weeks post-surgery. Surgery on the left side was performed six weeks later. Thus, the samples of the left side were evaluated after a healing time of three weeks. All samples were harvested after sacrifice for the purpose of showing an early stage of healing after three weeks (left humerus, femur, metacarpus, metatarsus) or a later healing time point nine weeks after surgery (right humerus, femur, metacarpus, metatarsus) (Fig. 1). Drill holes were left empty, filled with different soft, non-load bearing scaffolds, and bone replacement materials. Additionally, some defects in eight of the twelve sheep were filled with an autologous, cancellous bone graft harvested from the right and the left iliac crest. Cancellous bone graft was used as a positive control in the evaluation of the different filling materials. The empty drill holes were used as a negative control. All animal experiments were conducted following national and international regulations for the care and use of laboratory animals and were approved by local authorities (approval number G0341/12, LaGeSo, Berlin, Germany).
With the animal positioned in lateral recumbency, the humerus was stretched and rotated externally. The skin incision began at the level of the greater tubercule following the curve of the humerus approximately seven cm distal. After dissecting the subcutaneous fascia the cranial border of the acromial part of the deltoid muscle was elevated. The k-wire was positioned 2.5 cm distal to the edge of the tuberculum majus and at a distance of 2 cm lateral to the crista tuberculi majoris and drilled in a medial orientation to the caput humeri in all animals. Due to this standardized procedure the exact location of the drill hole in the small region of dense trabecular bone of the epimetaphysis of the proximal humerus was guaranteed. The drill hole was performed as described for the femur. A screw was placed as a landmark at a distance of two cm towards the shaft region of the humerus as described for the femur. The surgical approach was closed by continuous sutures in the detoideus muscle and subcutaneous tissue, and with simple interrupted sutures in the skin.
With the sheep lying in lateral recumbency, a skin incision was made over the lateral epicondyle of the humerus. The subcutaneous and deep fasciae along the cranial margin of the lateral head of the triceps muscle were incised and the lateral collateral ligament was identified on the lateral epicondyle. The drill hole was placed exactly in the center of the lateral epicondylus by the same procedure as used for the proximal femur. A screw was placed as a landmark in a distance of two cm towards the shaft region of the humerus. Both fasciae, the lateral collateral ligament, and the subcutaneous tissue were sutured continuously, while the skin was closed with simple interrupted sutures.
The surgical approach to the distal femur was performed from the medial side and therefore the animal had to be repositioned into dorsal recumbency, after all drill holes with a lateral approaches had been performed. The patella, tibia plateau and medial femur condyle are palpable. Skin incision was performed longitudinally following the curve of the femur between this landmarks. The underlying fascia was dissected and the cranial margin of the cranial belly of the sartorius muscle was incised with a scissor and cautery and retracted caudally. Then the caudal margin of the vastus medialis muscle was elevated to expose the underlying bone of the distal epimetaphyseal region. The bone surface was freed from periosteum with the raspatorium. The drill hole was placed proximal to the insertion of the medial collateral ligament. The small screw was inserted 2 cm proximal to the drill hole in direction to the shaft, as described for the other drill holes. The sartorius muscle and the fascia were closed, followed by the subcutaneous and skin suture.
Proximal-lateral and distal-medial metacarpal and metatarsal bones
The diaphyseal unicortical drill holes were placed in the proximal and distal parts of the left and right metacarpal and metatarsal bone. The proximal drill holes were placed from lateral, whereas the distal drill holes were performed from medial after repositioning of the sheep to the reverse side. The drill holes were placed exactly at the border of the first to second proximal quarter and the third and fourth quarter of the metacarpus and metatarsus. For a standardized positioning, the bone length was determined with a fluoroscope before sterile coverage of the sheep and the exact positions of the drill holes were marked with a surgical staple in the skin.
A 2 cm longitudinal incision was made through the skin, subcutaneous tissue and periosteum to the bone, taking care to avoid the digital extensor tendons. The k-wire was placed in the center of the bone and drilled transversely through one cortex (Fig. 3a). The unicortical drill hole was enlarged to 6 mm using the same procedure as for the epimetaphyseal bones (Fig. 3b). The second smaller drill hole for the locating screw was placed towards the shaft (Fig. 3c,d,f). The drill hole was cleaned of any periosteum, flushed with saline solution and dried with sterile gauze sponges (Fig. 3c). After defect filling, a thin layer of connective tissue and the subcutaneous skin were closed over the drill defect with continuous, absorbable sutures. The skin was closed with simple interrupted sutures of non-absorbable suture material.
The sheep were observed untill their recovery from general anesthesia. Immediately after surgery they were housed in small groups without restriction to movement. Clinical examination was performed daily. Postoperatively the sheep received a 75 μg/h fentanyl patch (Durogesic® 75 μg/h, Jannsen-Cilag GmbH, Neuss, Germany), which was renewed after three days and a dose of 2.2 mg flunixin-meglumin (Finandyne®, Intervet Deutschland GmbH, Unterschleißheim, Germany) per kg body mass daily for the first seven days after surgery. First day after surgery the sheep received additionally 500 mg metronidazol and 120 mg pantoprazole intravenously. 8.000 IE Dihydrostreptomycinsulfat, 4.8 mg Benzylpenicillin- Procain, and 3 mg Benzylpenicillin-Benzathin (Veracin®, Albrecht GmbH, Aulendorf, Germany) per kg body weight were given every second day until the skin sutures were removed.
All statistical analyses were performed using SPSS 20.0 Programm SPSS® Version 20 (SPSS GmbH Software, München, Germany). A p-value of <0.05 was considered as significant. A univariate ANOVA was performed. Data are presented in box plots showing the median, 25 and 75 percentile and min–max (whiskers).
Biomechanical in vitro testing
Biomechanical torsional test to failure was performed in order to investigate the stability of the bones after placing the diaphyseal drill holes and to ensure that the normal physiological strains do not affect the bones to a point of failure.
Comparison native MC vs. native MT bones
Comparison native vs. drilled MC bones
Comparison native vs. drilled MT bones
The comparison of the native MT and drilled MT (MTd) bones showed no statistical significant difference in torsional stiffness (Fig. 7a). The drilled MT bone showed a statistical significant decreased of the maximum torsional moment (p = 0.023), angle at Mmax (p = 0.016), and energy to failure (p = 0.014) (Fig. 7b-d). The two drill holes reduced the mean torsional stiffness by 13 %, the Mmax by 26 %, the mean angle at Mmax by 18 %, and the mean energy until failure by 39 %.
Comparison drilled MC vs. drilled MT bones
In summary, the native MC and MT bones showed no statistical significant difference in torsional stiffness but in all other measured parameters (Fig. 6a-d). Prepared with two 6 mm drill holes, only the bones of the hind legs (MT) showed a statistical significant decrease of the maximum torsional moment, angle at Mmax, and energy to failure, ranging in the same value as the bones of the front legs (MC; Fig. 7a-d). Between the drilled bones of the front and hind legs no statistical significant difference could be observed in any measured parameter.
In vivo results
All 13 sheep recovered from surgery and anesthesia without any complications. One animal had to be sacrificed after seven weeks due to a fracture of the left metacarpus one week after the second surgery. The other twelve animals survived the observation time of nine weeks without any complications. The general condition and physiological behavior was not affected by the surgery, so they were left in their group at all times. Only a minimal mean weight loss of 3 % and 0.5 % after the first and second surgery was registered over the course of time. All animals showed a declining moderate to slight degree of lameness in the limbs undergoing surgery until day seven post-surgery. The additional harvesting of cancellous bone graft from each iliac crest did not appear to have any impact on the severity of lameness. One animal developed a severe lameness of the left forelimb between day 10 to 21 post surgery, which was treated with a daily dose of 2.2 mg flunixin-meglumin (Finadyne®, Intervet Deutschland GmbH, Unterschleißheim, Germany), 8.000 IE Dihydrostreptomycinsulfat, 4.8 mg Benzylpenicillin- Procain, and 3 mg Benzylpenicillin-Benzathin (Veracin®, Albrecht GmbH, Aulendorf, Germany) per kg body weight every second day until recovery.
Radiographic analysis was performed for the documentation of the surgery result, as a support for sample explantation and a broad characterization of the healing result after 3 or 9 weeks of healing.
Epimetaphyseal drill holes of proximal and distal femur and humerus were in general more difficult to detect on in the radiographic images than diaphyseal drill holes, due to the surrounding trabecular bone. Three weeks after surgery the drill hole defects showed a slightly more radiodense zone surrounding the defect and mineralized callus formation from the edges towards the center of the drill hole (Fig. 4a,c,e,g). After nine weeks the sourrounding radiodense zone enlarged and the mineralized callus formation within the defect had progressed (Fig. 4b,d,f,h).
The drill holes of the left metacarpus and metatarsus were clearly detectible on the micro-radiographic images three weeks after surgery. The cortical boundary was sharp (Fig. 5a,c). Whereas the diaphyseal drill holes of the right metacarpus and metatarsus showed an advanced healing result with a strong bony mineralized callus formation from the cortex towards the center of the drill hole nine weeks after surgery (Fig. 5b,c).
The results indicate an advanced healing result for the diaphyseal drill holes after 9 weeks of healing and in general the difficulty to detect epimetaphyseal drill holes after a healing time of 9 weeks and the necessity of a reference screw.
With this study we introduce a new large animal model for the investigation of epimetaphyseal and diaphyseal drill hole defect regeneration in sheep. To the authors knowledge, a preclinical large animal model in which cancellous and cortical defect healing involving intramembraneous ossification can be investigated has not yet been published. Optionally two different healing time points can be analyzed in the same animal. There are several small and large animal osteotomy models for the investigation of a critical or non-critical sized cortical bone defects established, which involve in uncritical defect sizes the process of endochondral ossification [2, 3, 14, 22]. For the investigation of the special healing pattern of epimetaphyseal defects, independent small and large animal osteotomy models are also established [6–8, 10–13, 23–25]. In these models, cancellous bone defects regenerate under stable biomechanical conditions directly through intramembraneous ossification almost without intermediary cartilage [6–8, 10, 11, 13, 23]. In comparison to these models with only one harvested sample per animal, drill hole models have the advantage of an easy set-up. They offer a good reproducibility, a low morbidity in comparison to critical size defect models, contain mostly higher sample numbers per animal, and are accepted by some authors as a healing model for a fracture gap with a stable fixation [1, 26]. There is only one small animal drill hole defect model from Monfoulet et al., which allows the investigation of cortical and cancellous bone regeneration but compared to a large animal model with small defect sizes of 1 mm in the mice femur .
The cancellous bone defects in the here presented model were performed in reference to the well-established drill hole defect models in epimetaphyseal bone of the proximal and distal part of the femur and humerus, albeit with some modifications and improvements [15–18]. The described surgery technique has been refined by the use of a k-wire guided drilling. Precise drilling of holes with the cannulated-headed reamer prevents a slippage of the large 6 mm drill and can avoid complications like fractures related to a failures during defect preparation as it has been described in the hitherto models . Additionally, for the precise explantation of samples a method for marking was introduced by the use of two custom-made templates and a small reference screw.
The position of the drill hole in the proximal femur was adapted to the dimensions of the larger black-faced sheep compared to the hitherto used Swiss alpine sheep. To ensure a precise placement in the rather small proximal, cancellous, epimetaphyseal end of the femur, the drill hole was placed in a distance of 2.5 cm from the distal tip of the major trochanter. Other drill hole defect models use the proximal epimetaphyseal part of the tibia, but due to the short area with stable cancellous bone, the tibia was not included in this model [27–29]. In addition, the approach to the distal femur was modified in our model and performed from the medial side to avoid the more pronounced muscle layer on the lateral side of the condyles.
For the investigation of diaphyseal drill hole defect regeneration, the metacarpal and metatarsal bones were chosen, according to their high similarity in shape and size and their general acceptance in cortical defect models . Petrizzi et al. used the metacarpal bone of the left side for a bicortical drill hole model for the investigation of the effect of naloxone on bone regeneration but without a biomechanical test of the metacarpal bone to evaluate the influence of the defect size on the bone . Other studies in sheep used the tibia for the placement of smaller single or multiple, uni- or bicortical drill hole defects [31, 32].
For our model, both unicortical defects per bone were placed in a maximal distance to each other in the proximal lateral and distal medial shaft region to have low mutually influential effects. The drill hole defect size was determined to 6 mm in diameter, to stay below the recommended defect sized of 50 % related to the diameter of the utilized metacarpal and metatarsal bones . The epimetaphyseal drill hole defect size was adapted to the diaphyseal drill hole size. Prior to the in vivo testing, a biomechanical torsion test until failure of native and drilled metacarpal and metatarsal bones of four test sheep were performed to ensure that our defect does not affect the bone stability in a way that would induce fractures during physiological load. The native MC bones of the front legs showed a significant lower maximum torsion moment until failure in comparison to the MT bones, as they are macroscopically slightly smaller and thinner. Nevertheless, defect drilling significantly decreased only the Mmax, angle at Mmax, and energy to failure of the MT bones compared to the corresponding healthy contralateral MT bones. The torsional stiffness, Mmax, angle at Mmax, and energy to failure of the drilled MT bones ranged after defect creation at the same level as the healthy and also the drilled MC bones. Therefore we concluded, that MC bones of the front legs and MT bones of the hind legs prepared with two 6 mm unicortical drill holes might be comparable in their biomechanical behavior. Biomechanical properties of the MT bones of the hind legs were more affected by the drill holes than the MC bones of the front legs. Influence of slightly different loading situation on defect healing might be normalized by a distribution of samples in a randomized complete block design, leading to an equalized number of samples from one material in the MC or MT bones. The calculated existing moments on the MT sheep bone in the musculoskeletal lower limb model with a high safety margin, were much lower during a normal gait cycle than the maximum torsion moments until failure. Therefore, we concluded that MC and MT bones prepared with two 6 mm unicortical drill holes might withstand the acting moments during healing time. Nevertheless, a failure cannot be completely excluded because one animal had to be sacrificed earlier due to a spiral fracture of the left MC bone. This shows that the existing peak moments in an experimental sheep stable without restriction to movement might range up to the level of the measured moments until failure. However, a failure rate of approximately 8 % for the diaphyseal drill holes seems to be tolerable, compared to the amount of 96 epimetaphyseal and 96 diaphyseal evaluable samples within 12 sheep, 8 of them with additional cancellous bone graft harvesting from the iliac crest. Particularly, as the sheep health status and behavior was only slightly affected during the study.
In our opinion, the combination of calculation of acting moments from the musculoskeletal sheep model and a previous biomechanical failure test proved to be an adequate technique for the estimation of acting forces and safety against failure of this new in vivo defect model. Investigations of promising biomaterials in this model focus on the stimulation and improvement of intramembranous bone healing under stable biomechanical conditions, but provide the possibility of a regular animal housing without restriction to movement like cast bandage or sling-system immobilization.
Nevertheless, this model is limited to the certain drill hole defect size and localizations and cannot replace critical-size defect studies or the investigation of biomaterials in the later targeted application site. Besides, sheep have cancellous and cortical bone and show a similar bone remodeling and turnover rate as humans, but have a plexiform bone structure with few Haversian canals unlike the human bone [33, 34]. As no single animal model completely recapitulates human bone regeneration each species provides advantages and limitations [35, 36], which need to be considered carefully before choosing the suitable animal for a drill hole model. In general, an extend of this drill hole model to other large animals as goats or pigs is conceivable. As goats have a comparable body weight and metabolic rate to humans [36, 37], they are commonly used as a model in bone defect [38, 39] and osteochondral defect studies [39, 40]. The huge weight of adult pigs with proportional short limbs could be challenging within this model , albeit mini-pigs might be suitable as they have been already used in studies to investigate osteointegration and bone defect healing [42, 43]. Nevertheless, the switch to another species would require further biomechanical evaluations for a feasible defect size. Additionally, it would be necessary to adapt the surgical approach as well as the observation time points to the respective animal species.
This new preclinical large animal drill hole model allows the simultaneous investigation of new bone tissue engineering strategies in cancellous and cortical bone defect healing sites. The sheep model combines the advantages of an adequate defect size for the targeted application in a patient and a sufficient number of samples per animal for the investigation of intramembranous bone defect regeneration. Beside that the combination of the calculation of acting moments from the musculoskeletal lower limb model and biomechanical in vitro testing proofed to be an adequate method to keep the accidental fracture risk low. This model is intended to be used as a simplified screening tool for the selection of promising biomaterials before an application in a complex treatment site.
This study was support by the Berlin-Brandenburg Center for Regenerative Therapies, Charité – Universitätsmedizin Berlin. We thank Elisabeth Huber at the Julius Wolff Institute for her assistance in the surgeries and animal care.
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