2.3. In Vivo Bone Healing Study

The bone regeneration potential of the different treatments was studied in a rat osteotomy model of delayed healing as previously described [36]. A total of 18 adult female Sprague-Dawley rats (aged >7 months, ≥300 g, Janvier Labs, Le Genest-Saint-Isle, France, that had more than three litters (ex-breeders) were included in this study. Rats were kept in small groups under obligatory hygiene standards and conventional housing conditions with controlled temperature set to 20 ± 2 °C, a light/dark period of 12 h and food and water being available ad libitum. All animal experiments were approved by the local animal protection authorities (Landesamt für Gesundheit und Soziales Berlin, Germany: G0258/18, 19 December 2018) and performed in accordance with the German Animal Welfare Act, the National Institutes of Health Guide for Care and Use of Laboratory Animals and the ARRIVE guidelines.

Before starting the surgery, the rats were anesthetized by inhalation of isoflurane (Forene, Abott, Wiesbaden, Germany) and received a potent analgesic (bubrenorphine, RB Pharmaceuticals, Berkshire, UK; 0.1 mg/kg BW), an antibiotic bolus (clindamycin, Ratiopharm, Ulm, Germany; 45 mg/kg BW) and eye ointment. The blood sampling and the osteotomy were carried out under deep anesthesia on a heating plate set to 37 °C.

To create the autologous blood clot, blood was drawn from the vena saphena of the right hind limp using a 3.2% sodium citrate-coated syringe. Then, 180 µL of blood was mixed with 0.75 mg SD-MBG contained in 6.25 µL of 0.9% NaCl solution and 7 µL thrombin (500 i.E./mL, 12% CaCl₂, Baxter, Deerfield, MA, USA) for the induction of coagulation. A cylindrical mold enabled standardization of the resulting shape.

The operation area of the left femur was clipped and disinfected, the femur was exposed by a longitudinal skin incision and blunt preparation of the muscles. An external fixator (RatExFix, RISytem, Davos, Switzerland) was mounted on the femur, followed by the creation of a 2 mm osteotomy using an oscillating saw (W&H, Bürmoos, Austria) and a saw guide. The coagulated, autologous blood clot containing 0.75 MBG ± BMP-2 load was inserted into the osteotomy gap. The wound was closed with sutures and the rats were returned to their cages. As post-operative analgesia, tramadolhydrochloride (Grünenthal, Aachen, Germany; 0.5 mg/mL) was added to the drinking water for three days post-surgery.

At 2 and 4 weeks post-surgery, the animals were radiologically examined by X-rays and in vivo micro-computed X-ray tomography (µCT) (for details, see section below) under anesthesia induced by intraperitoneal injection (i.p.) of ketamine hydrochloride (Actavis Switzerland, Regensdorf, Switzerland; 60 mg/kg BW) and medetomidine (CP-Pharma, Burgdorf, Germany; 0.3 mg/kg BW). To recover from anesthesia, an antidote (1.5 mg/kg BW atipamezol, CP-Pharma, Burgdorf, Germany) was injected intra-muscularly (i.m.) from the 2 week time point. The final study time point was set to 4 weeks post-surgery. After in vivo radiological examinations, blood was collected by intracardiac puncture, the animals were euthanized under deep anesthesia by intracardiac injection of potassium chloride, and the osteotomized femur was harvested. The bones were fixed in 4% paraformaldehyde (PFA; Science Services, München, Germany) in PBS for 24 h at 4 °C. The bones were again imaged using a higher resolution µCT, and afterwards decalcified, dehydrated and paraffin-embedded for histological analysis.

At 2 and 4 weeks post-osteotomy, the animals were radiologically examined by X-rays and in vivo µCT (Viva 40, SCANCO Medical, Wangen-Brüttisellen, Switzerland) under anesthesia. µCT nominal resolution was set at 35 µm voxel size, with 55 kV source voltage and 145 µA source current. A global threshold was applied to all bones corresponding to a bone mineral density of 408 mg/cm³ calcium hydroxyapatite (CaHA). Four weeks post-osteotomy, bones were harvested and cleaned of excess of soft tissue, fixed in 4% PFA/PBS for 24 h and rinsed thoroughly in PBS. Harvested bones were mechanically fixed within a radiologically transparent serological pipette to keep the integrity of the bone and immersed in PBS. µCT scans were then performed using a Bruker SkyScan 1172 high-resolution micro-CT (Bruker, Kontich, Belgium) with a nominal resolution of 8 µm, 0.5 mm aluminum filter, 80 kV source voltage and 124 µA source current. A camera pixel binning of 2 × 2 was used together with an orbital scan of 180 degrees in steps of 0.3 degrees. Reconstruction was performed using the SkyScan NRecon software. Gaussian smoothing, ring artifact reduction, misalignment compensation and beam hardening correction were applied. The volume of interest (VOI) was defined to include the 2 mm defect region and 1 mm in the proximal and distal direction from the cutting plane of the bone defect. A global threshold was applied to all bones corresponding to a bone mineral density of 435 mg/cm³ CaHA. Calibration was performed using phantoms containing 0.25 and 0.75 g/cm³ CaHA (Bruker, Kontich, Belgium) homogenously distributed in epoxy rods of similar diameter as of the scanned bones to minimize beam hardening error.

Decalcification of bones occurred over 4 weeks at 37 °C using decalcifier soft solution (Carl Roth, Karlsruhe, Germany) and changing the decalcifying solution twice per week, followed by de-hydration and paraffin-embedding. Paraffin-embedded bones were sectioned into 5 µm-thick sections, de-paraffinized by 2 × 10 min of incubation in xylol and re-hydrated by descending alcohol series and distilled water as the final step before staining, stained with MOVAT’s pentachrome as previously described [37] and with sirius red (Sigma-Aldrich, St. Louis, MI, USA). Briefly, slides were incubated for 1 h in 1% sirius red solution and washed twice with 0.5% acetic acid (Sigma-Aldrich, St. Louis, MI, USA). Afterwards, slides were washed in 1% acetic acid. At the end of the staining, the slides were dehydrated using xylol (Fisher Chemical, Thermo Fisher Scientific, Waltham, MA, USA) and embedded with Vitroclud (Langenbrink, Emmendingen, Germany).

For the immunohistochemical analysis, after deparaffinization and re-hydration, slides were blocked using 5% normal horse serum (Vector Laboratories, Burlingame, CA, USA) for 1 h and 1% bovine serum albumin (BSA)/PBS, followed by overnight incubation at 4 °C with anti-alpha-smooth muscle actin (α-α-SMA) (1:400, mouse monoclonal, clone 1 A4, DAKO Agilent Technologies, Santa Clara, CA, USA) or anti-cluster of differentiation 68 (α-CD68) (1:2000, mouse monoclonal, clone BM4000, OriGene Technologies, Rockville, MD, USA). An α-mouse, rat adsorbed biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA) diluted 1:50 in 2% normal serum horse and 1% BSA/PBS was incubated on the slides for 30 min. AB complex (Vector AK 5000, Vector Laboratories, Burlingame, CA, USA) was incubated for 50 min, then the milieu was slightly alkalized by using a chromogen buffer (pH 8.2), followed by the visualization of the staining (Vector SK 5100, Vector Laboratories, Burlingame, CA, USA). As counterstaining, hematoxylin (Mayer’s) was chosen and the slides were embedded using Aquatex (Merck, Darmstadt, Germany). Microscopic images of all slides were taken at 10× magnification under bright field (Axioskop 40, Carl Zeiss, Oberkochen, Germany). Histomorphometric analyses were carried out using the MOVAT’s pentachrome-stained slides and applying a custom-made macro embedded in FIJI ImageJ Software [35]. The callus area was determined manually. Detection of mineralized tissue and cartilage was performed according to color thresholding to determine the areas of the respective tissues. Blood vessels and osteoclasts were revealed in an analog manner by α-SMA and CD68 staining, respectively. Finally, the amount of blood vessels was normalized to the total area of the callus. The length of mineralized callus surface covered by CD68+ cells to was normalized to the total length/perimeter of the mineralized callus.