## 2 Materials and Methods
### Animal Experiment Surgery Procedure and Study Design  
The experimental procedures were approved by the Ethical Committee of the Faculty of Veterinary Medicine, University of Belgrade and the Ministry of Agriculture, Forestry and Water Management of the Republic of Serbia (No. 323-07-08477/2015-05/3), and they  
Figure 1: Experimental setup: (**a**) schematic of the surface dielectric barrier discharge (SDBD) system and (**b**) voltage and current waveforms.  
were performed in accordance with Directive 2010/63/EU. Ten four-month-old Chinchilla rabbits weighing between 3.5 kg and 4.3 kg were used in this study.  
Enroffloxacin (Baytril 2.5%, Bayer, Leverkusen, Germany) 10 mg/kg i.m. was used in the premedication and on five postoperative days, as well as butorphanol (Butomidor 10 mg/mL, Richter Pharma Ag, Wels, Austria) 0.5 mg/kg s.c., for pain management.  
In all the animals, general anesthesia was induced via intramuscular injection of 35 mg/kg of ketamine hydrochloride (Ketamidor 10%, Richter Pharma AG, Wels, Austria) and 5 mg/kg of xylazine (Xylased 20 mg/mL, Bioveta, Ivanovice na Hane, Czech Republic). The midline incision of the scalp, created between the base of the ears and approximately 5 cm anteriorly, was performed for the full-thickness skin flap. The parietal bones were exposed after sharp subperiosteal dissection of the pericranium. Four full-thickness calvarial critical-sized defects, measuring 8 mm in diameter, were created via electrical drilling under copious saline irrigation, two of them on each side of the midline of the parietal bone (Figure 2a). A surgical template made of a sterile acrylic disk was used to ensure each defect was identical.  
In every rabbit, each of the four defects was randomly allocated to one of the following study groups:  
Group I--defect filled with beta TCP.  
Group II--defect filled with beta TCP after treatment with cold atmospheric plasma.  
Group III--defect filled with beta TCP in combination with PDLSC.  
Group IV--defect filled with beta TCP after treatment with cold atmospheric plasma in combination with PDLSC.  
Although additional control groups would have added value to the study, we were limited by the fact that the rabbit calvaria can fit only up to four critically sized defects. In  
Figure 2: Calvarial critical-sized defects of 8 mm of diameter in a rabbit model: (**a**) before and (**b**) after implantation.  
this study, we opted for \(\beta\)-TCP as a base control, since it is graft material that is already used for the treatment of bone defects and has been proven to be effective.  
The bone substitutes filled the bone defects from the dura to the level of marginal bone, replacing the volume of the removed bone, and the pericranium and skin were sutured in layers with absorbable sutures (Figure 2b). The surgical wound was treated once with Engomycin Spray (Intervet Productions S.r.I., Aprilia, Italy). During the postoperative animal care, the rabbits were observed twice a day until the end of the experiment. The sutures were removed 12 days after the surgery. For pain assessment, the Rabbit Grimace Scale of the National Center for the Replacement, Refinement and Reduction of animals in the research scale of Newcastle University was used.  
One rabbit died postoperatively, while the remaining animals (five at two weeks and four at four weeks postoperatively) were anesthetized according to the protocol and then euthanized via intravenous application of 150 mg/kg sodium pentobarbitone. The calvarial bones were surgically removed and separated into different bone blocks according to the study groups.