In vivo experiments

3.1.1. Non-critical defect femoral osteotomy model

For the in vivo animal studies female three and twelve months old ex-breeder Sprague Dawley rats from Charles River WIGA Deutschland GmbH were used. This strategy was applied following previous studies from Preininger [194] and Strube [195], where it was shown that aged female rats, after a minimum of three litters, typically displayed a non-union over the time course of six weeks, when receiving a 2 mm osteotomy gap in the left femoral midshaft. This serves as a model for biologically impaired fracture healing and stands in contrast to the three months old rats, showing a complete fracture union after the same amount of time. The aged cohort (twelve months old animals that have had a litter of at least three) was kept at the “Tierexperimentelle Einrichtung der Charité Berlin, des Campus Virchow Klinikum” for aging, where they lived an artificial light-induced day-night rhythm of twelve hours at constant temperature (21°C) and humidity (60-70%). Diet contained dry pellets and water, which was consumed according to own needs. Animal experiments were conducted in compliance with the ARRIVE guidelines and according to the policies and principles of the Animal Welfare Act, the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the National Animal Welfare Guidelines. All animal experiments were approved by the local legal representative (Institutional Animal Care and Use Committees, LaGeSo, G0120/14, G0172/15).

3.1.2. External fixation

The external fixation system used for this work comprised a steel fixation bar and 4 titanium clampy, which are fixated by head screws and locknuts at each side. The external fixateur is placed unilaterally on the left femoral bone with 4 titanium kirschner wires, each 1.23 mm in diameter (Figure 10A). The distance between the outer and inner wire on each side is 4 mm, while there is a 10 mm distance between the inner wires, where a standardized 2 mm fracture gap was introduced. This was achieved by the help of a sawing template, placed on the wires and the fixateur (Figure 10B). The fixateur bar was always placed in the same distance from the left leg by using a plastic block of 7.5 mm height. A sketch of the assembled and fixated system stabilizing the osteotomized femur is found in Figure 10C.

3.1.3. Surgical procedure

All animals included in the study, were assessed for their overall health and body weight. In preparation for the surgical procedure animals received 20 mg Tramadol per kilogram body weight, as an anti-pain agent. Animals were anesthetized with 0.3 mg/kg Medetomidin Domitor and 60 mg/kg Ketamin by intraperitoneal injection prior to surgical procedure. To prevent infection, 45 mg/kg Clindamycin was administered by subcutaneous injection and eyes were prevented from drying out by application of Bepanthen® eye balm. A longitudinal skin incision was made over the left femur. The bone was exposed by blunt fascia dissections. Unilateral external fixators were used to stabilize the fracture, made of stainless steel and titanium. For an exact placement of the four wire holes (diameter: 1 mm), a drilling template was used for every procedure. The first wire hole was placed by optical assessment, vertical to the femur orientation, at the distal side of the femur midshaft (diaphysis). It was drilled monocortically and the titanium wire was carefully fitted. The drilling stencil for the remaining wire holes was placed onto the first titanium wire. All other holes were drilled bicortically and the wires placed respectively. After incision of the titanium wires, the drilling template was exchanged for an external fixator bar and slightly fastened by screws with a 7.5 mm distance from the femoral bone. Using a sawing stencil to accomplish maximal reproducibility and standardization, the femur was double osteotomized with a width of 2 mm within the inner titanium wires. Saline solution was used throughout the procedure preventing unwanted tissue damage by heat induction. Muscle fascia and skin were closed using absorbable and non-absorbable sutures, respectively. Animals received 1.5 mg/kg Antisedan, an anesthetic antagonist, and were placed under red light until awakening. Animals were kept in cages of two after the surgical procedure to minimize wound nibbling and scratching and thereby disturbing the progression of healing. They received Tramadol as an anti-pain agent

[195]

[194, 195].

Figure 10. External fixation system for osteotomized rat femurs.

Custom made external rat femoral fixator after Strube et al. was used for this work. (A) Assembled steel fixator with bar, screws, nuts and titanitum Kirschner wires. (B) Sawing template for 2 mm osteotomy (C) Image of external fixator holding the dissected osteotomized femoral bone with remaining muscle tissue. Pictures adapted from Strube et al. and Preininger et al.

though the drinking water for three days post-surgery. Throughout the whole period, animals were monitored for health, behavioral contentment and wound healing. Fracture healing was assessed after 3, 7 and 14 days by euthanization and femur dissection.

3.1.4. Sacrifice and specimen harvest

Animals were sacrificed at day 3, 7 and 14 post-surgery. They were administered 0.3 mg/kg Medetomidin and 60 mg/kg Ketamin for anaesthesia by intraperitoneal injection. For serum isolation, 5-10 ml blood was drawn by intracardial punctuation and later centrifuged at 2.000 x g for 10 minutes to separate blood cells and clotting components from the serum. Aliquots were stored at -80°C until further usage. Animal sacrifice was performed by intracardiac injection of 7 ml potassium chloride (KCl) solution (1 M), inducing cardiac arrest. The operated left femur was dissected and dislodged from the joints, followed by gentle removal of the surrounding and remaining muscle tissue. Depending on the down-stream analysis, different protocols were carried out:

1) For Ribonucleic acid (RNA), metabolite and protein analysis, the hematoma was extracted from the osteotomy gap, together with 1 mm of the neighboring proximal and distal tissue (region of interest), placed in cryo vials and snap frozen in liquid nitrogen and stored in nitrogen tanks until further processing (see chapter 3.2.). 2) For ex vivo cytokine explant cultures, the hematoma was carefully extracted from the

osteotomy gap, together with 1 mm of the neighboring tissue from distal and proximal. Each specimen was weighted and covered with a respective amount of prewarmed media before culturing (see chapter 3.3.).

3) For histological analysis, femurs were placed in 4% paraformaldehyde (PFA)/phosphate buffered saline (PBS) for tissue fixation. External fixateurs were not removed until embedding commenced. Condyles on the distal and proximal sides were incised to ensure full tissue penetration of the fixating solution (see chapter 3.4.).