Romano Matthys

Fracture healing in mice under controlled rigid and flexible conditions using an adjustable external fixator.

Mice are increasingly used to investigate mechanobiology in fracture healing. The need exists for standardized models allowing for adjustment of the mechanical conditions in the fracture gap. We introduced such a model using rigid and flexible external fixators with considerably different stiffness (axial stiffnesses of 18.1 and 0.82 N/mm, respectively). Both fixators were used to stabilize a 0.5 mm osteotomy gap in the femur of C57BL/6 mice (each n = 8). Three‐point bending tests, µCT, and histomorphometry demonstrated a different healing pattern after 21 days. Both fixations induced callus formation with a mixture of intramembranous and enchondral ossification. Under flexible conditions, the bending stiffness of the callus was significantly reduced, and a larger but qualitatively inferior callus with a significantly lower fraction of bone but a higher fraction of cartilage and soft tissue was formed. Monitoring of the animal movement and the ground reaction forces demonstrated physiological loading with no significant differences between the groups, suggesting that the differences in healing were not based on a different loading behavior. In summary, flexible external fracture fixation of the mouse femur led to delayed fracture healing in comparison to a more rigid situation.

Development of a stable closed femoral fracture model in mice.

BACKGROUND Mice have become of increasing interest as experimental model for fracture studies. Due to their small size, most studies use simple pins for fracture stabilization, although insufficient rigidity of fixation critically affects fracture healing. Herein, we studied whether longitudinal fracture compression by an intramedullary screw represents a standardized, stable osteosynthesis technique in mice, and whether it may accelerate fracture healing. MATERIALS AND METHODS A micro-screw (MouseScrew) was constructed, allowing closed fracture stabilization without traumatizing surgery. Fracture stabilization was achieved by longitudinal compression, which was confirmed by biomechanical testing of osteotomized cadaver femora. Bone repair was analyzed histomorphometrically at 2 and 5 wk after surgery. RESULTS Ex vivo analyses showed a significantly increased rotational and axial stiffness after screw stabilization (n = 8 each) compared with stabilization techniques using a conventional pin (n = 8 each) or a locking nail (n = 8 each). In the in vivo setting, 2 wk of screw stabilization (n = 8) demonstrated a significantly decreased fibrous tissue formation and an increased cartilage production compared with fractures stabilized by the locking nail (n = 8). After 5 wk callus consisted exclusively of bone in all animals studied without differences between the two stabilization techniques (n = 8 each). CONCLUSIONS Because prolonged fibrous tissue formation indicates delayed fracture healing, we conclude that the increased stability of the fracture by the use of our newly developed MouseScrew accelerates initial bone repair. Further, this fracture model may represent an ideal tool to study bone repair in mice under conditions of stable fixation.

Fixation compliance in a mouse osteotomy model induces two different processes of bone healing but does not lead to delayed union

Delayed unions are a problematic complication of fracture healing whose pathophysiology is not well understood. Advanced molecular biology methods available with mice would be advantageous for investigation. In humans, decreased fixation rigidity and poor reduction are generally associated with delayed unions. In this study, these two factors were combined to observe their effect on bone healing in mice. Two plates with locking screws, one with 14 the bending stiffness of the other, were used to stabilize a 0.45mm gap osteotomy. muCT, radiographs, 4pt-bending tests and histological analysis demonstrated that the different plate types led to two different healing pathways. The less flexible bridging plate induced only intramembranous ossification whereas the more flexible bridging plate induced a mixture of endochondral and intramembranous ossification. However, the different plates led to a delay in healing of only 3-5 days in the period between 14 and 21 post-operative days. In mice, considerable fixation flexibility is necessary to induce secondary bone healing similar to that which occurs in humans, but this was not sufficient to induce a substantial delay in bone healing as would be expected in humans.

An internal locking plate to study intramembranous bone healing in a mouse femur fracture model.

In most murine fracture models, the femur is stabilized by an intramedullary implant and heals predominantly through endochondral ossification. The aim of the present study was to establish a mouse model in which fractures heal intramembranously. Femur fractures of 16 SKH‐mice were stabilized by an internal locking plate. Femur fractures of another 16 animals were stabilized by an intramedullary screw. Bone repair was analyzed by radiographic, biomechanical, and histological methods. At 2 weeks, histological analysis showed a significantly smaller callus diameter and callus area after locking plate fixation. Cartilage formation within the callus could only be observed after screw fixation, but not after fracture stabilization with the locking plate. Radiological and biomechanical analysis after 2 and 5 weeks showed a significantly improved healing and a higher bending stiffness of fractures stabilized by the locking plate. Fractures stabilized by the locking plate healed exclusively by intramembranous ossification, which is most probably a result of the anatomical reduction and stable fixation. The fractures that healed by intramembranous ossification showed an increased stiffness compared to fractures that healed by endochondral ossification. This model may be used to study molecular mechanisms of intramembranous bone healing.

Ex vivo analysis of rotational stiffness of different osteosynthesis techniques in mouse femur fracture

The various molecular mechanisms of cell regeneration and tissue healing can best be studied in mouse models with the availability of a wide range of monoclonal antibodies and gene‐targeted animals. The influence of the mechanical stability of individual stabilization techniques on the molecular mechanisms of fracture healing has not been completely elucidated yet. Although during recent years several osteosynthesis techniques have been introduced in mouse fracture models, no comparative study on fracture stabilization is available yet. We therefore analyzed herein in a standardized ex vivo setup the rotational stiffness of seven different osteosynthesis techniques using osteotomized right cadaver femora of CD‐1 mice. Uninjured femora without osteotomy served as controls. Femur stabilization with a locking plate or an external fixator resulted in a rotational stiffness almost similar to the intact femur. The use of a “pin‐clip” device, a “locking nail,” a “mouse nail,” or an “intramedullary screw” produced a lower torsional stiffness, which, however, was still significantly higher than that achieved with the widely applied conventional pin. By the use of the presented data a more specific choice of stabilization technique will be possible according to the various questions concerning molecular aspects in fracture healing.

Melatonin Impairs Fracture Healing by Suppressing RANKL-Mediated Bone Remodeling

BACKGROUND Melatonin, the major pineal hormone, is known to regulate distinct physiologic processes. Previous studies have suggested that it supports skeletal growth and bone formation, most probably by inhibiting bone resorption. There is no information, however, whether melatonin affects fracture healing. We therefore studied in a mouse femur fracture model the influence of melatonin on callus formation and biomechanics during fracture healing. METHODS AND MATERIALS Thirty CD-1 mice received 50 mg/kg body weight melatonin i.p. daily during the entire 2-wk or 5-wk observation period. Controls (n = 30) received equivalent amounts of vehicle. Bone healing was studied by radiological, biomechanical, histomorphometrical, and protein biochemical analyses at 2 and 5 wk after fracture. RESULTS Biomechanical analysis at 2 wk after fracture healing showed a significantly lower bending stiffness in melatonin-treated animals compared with controls. A slightly higher amount of cartilage tissue and a significantly larger callus size indicated a delayed remodeling process after melatonin treatment. Western blot analysis showed a significantly reduced expression of receptor activator of nuclear factor-κB ligand (RANKL) and collagen I after melatonin treatment. The reduced expression of RANKL was associated with a diminished number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts within the callus of the newly formed bone. CONCLUSIONS Because bone resorption is an essential requirement for adequate remodeling during fracture healing, we conclude that melatonin impairs fracture healing by suppressing bone resorption through down-regulation of RANKL-mediated osteoclast activation.

Sildenafil accelerates fracture healing in mice

Sildenafil, a cyclic guanosine monophosphate (cGMP)‐dependent phospodiesterase‐5 inhibitor, has been shown to be a potent stimulator of angiogenesis through upregulation of pro‐angiogenic factors and control of cGMP concentration. Herein, we determined whether sildenafil also influences angiogenic growth factor expression and bone formation during the process of fracture healing. Bone healing was studied in a murine closed femur fracture model using radiological, biomechanical, histomorphometric, and protein biochemical analysis at 2 and 5 weeks after fracture. Thirty mice received 5 mg/kg body weight sildenafil p.o. daily. Controls (n = 30) received equivalent amounts of vehicle. After 2 weeks of fracture healing sildenafil significantly increased osseous fracture bridging, as determined radiologically and histologically. This resulted in an increased biomechanical stiffness compared to controls. A smaller callus area with a slightly reduced amount of cartilaginous tissue indicated an accelerated healing process. After 5 weeks the differences were found blunted, demonstrating successful healing in both groups. Western blot analysis showed a significantly higher expression of the pro‐angiogenic and osteogenic cysteine‐rich protein (CYR) 61, confirming the increase of bone formation. We show for the first time that sildenafil treatment accelerates fracture healing by enhancing bone formation, most probably by a CYR61‐associated pathway.

The LockingMouseNail—A New Implant for Standardized Stable Osteosynthesis in Mice

BACKGROUND Mouse models are of increasing interest to study cellular and molecular mechanisms during fracture healing. However, unlike in large animals and in humans, stable fixation of fractures has been difficult due to the small size of the mouse. METHODS Based on μCT-scans of a mouse femur, we developed a new intramedullary implant system comparable to a human locking nail. We analyzed fracture healing with osteotomy gap sizes of 0.00, 0.25, and 2.00 mm, which were stabilized with the LockingMouseNail. RESULTS Femora with a gap size of 0.00 mm and 0.25 mm showed complete fracture healing after 5 wk. Femora showed a secondary bone healing pattern with induction of a small periosteal callus. In contrast, femora with a gap size of 2.00 mm showed sparse periosteal callus formation and a lack of bone bridging even after 10 wk, indicating atrophic non-union. CONCLUSION The LockingMouseNail allows standardized fixation of mouse femur fractures and also stabilization of segmental defects. By introducing different gap sizes, the healing process can be influenced, ranging from normal fracture healing to atrophic non-union formation. Therefore, the model may ideally be suited to study molecular mechanisms of normal fracture healing, delayed healing, and non-union formation. It may additionally allow studying biological properties and effectiveness of different bone substitutes in stabilized segmental defects.

Mechanical evaluation of a new minimally invasive device for stabilization of proximal humeral fractures in elderly patients A cadaver study

Background Treatment of proximal humerus fractures in elderly patients is challenging because of reduced bone quality. We determined the in vitro characteristics of a new implant developed to target the remaining bone stock, and compared it with an implant in clinical use. Methods Following osteotomy, left and right humeral pairs from cadavers were treated with either the Button-Fix or the Humerusblock fixation system. Implant stiffness was determined for three clinically relevant cases of load: axial compression, torsion, and varus bending. In addition, a cyclic varus-bending test was performed. Results We found higher stiffness values for the humeri treated with the ButtonFix system—with almost a doubling of the compression, torsion, and bending stiffness values. Under dynamic loading, the ButtonFix system had superior stiffness and less K-wire migration compared to the Humerusblock system. Interpretation When compared to the Humerusblock design, the ButtonFix system showed superior biomechanical properties, both static and dynamic. It offers a minimally invasive alternative for the treatment of proximal humerus fractures.

A new model to analyze metaphyseal bone healing in mice.

BACKGROUND Despite the increasing clinical problems with metaphyseal fractures, most experimental studies investigate the healing of diaphyseal fractures. Although the mouse would be the preferable species to study the molecular and genetic aspects of metaphyseal fracture healing, a murine model does not exist yet. Using a special locking plate system, we herein introduce a new model, which allows the analysis of metaphyseal bone healing in mice. METHODS In 24 CD-1 mice the distal metaphysis of the femur was osteotomized. After stabilization with the locking plate, bone repair was analyzed radiologically, biomechanically, and histologically after 2 (n=12) and 5 wk (n=12). Additionally, the stiffness of the bone-implant construct was tested biomechanically ex vivo. RESULTS The torsional stiffness of the bone-implant construct was low compared with nonfractured control femora (0.23 ± 0.1 Nmm/°versus 1.78 ± 0.15 Nmm/°, P<0.05). The cause of failure was a pullout of the distal screw. At 2 wk after stabilization, radiological analysis showed that most bones were partly bridged. At 5 wk, all bones showed radiological union. Accordingly, biomechanical analyses revealed a significantly higher torsional stiffness after 5 wk compared with that after 2 wk. Successful healing was indicated by a torsional stiffness of 90% of the contralateral control femora. Histological analyses showed new woven bone bridging the osteotomy without external callus formation and in absence of any cartilaginous tissue, indicating intramembranous healing. CONCLUSION With the model introduced herein we report, for the first time, successful metaphyseal bone repair in mice. The model may be used to obtain deeper insights into the molecular mechanisms of metaphyseal fracture healing.