Samples preparation
Chitosan (CTS; low molecular weight; Sigma-Aldrich, shrimp sourced) was used for the studies. Gallic acid (GA), ferulic acid (FA), and tannic acid (TA) were purchased from Sigma-Aldrich company. Chitosan, tannic acid, gallic acid, and ferulic acid were dissolved in 0.1M acetic acid at 1% concentration separately. Chitosan solution was mixed with 10 (w/w%) addition of phenolic acid solution for 1h. The mixtures were then placed in the 24-well sterile cell culture plates (2.5 mL/well), frozen for 24 h in -18oC, and lyophilized (ALPHA 1–2 LDplus, CHRIST,−20°C, 100 Pa, 48 h). As a result, the three-dimensional dry samples (scaffolds) were obtained.
In Vivo Experiment
The in vivo experiment was carried out on a group of male New Zealand rabbits weighing 2.8–3.2 kg. The rabbits were purchased at the Experimental Medicine Center of the Medical University of Silesia in Katowice, fak. No.KCM / FPS / 0041/06/20. Before the procedure, the animals' health was checked. The animals were under constant veterinary supervision and were given a vaccine: Castomix by Pharmagal Bio against Myxomatosis (MXT) and rabbit haemorrhagic disease (RHDV). All research protocols were approved by the Local Ethics Committee of the University of Life Sciences in Lublin No. 104/2017, and the experiment was conducted in accordance with the provisions on animal protection. The animals were placed in the animal facility of the Experimental Medicine Center of the Medical University of Lublin. During this time, their natural habits were monitored and the temperature of each animal was measured daily. The general condition of the rabbits was very good, with no clinical signs of disease. The daily measured body temperature was within the reference range.
For 7 days after the herd was introduced to the Vivarium, their body temperature was measured, the food intake and the behavior of the animals were observed. After a week's adaptation, the rabbits were prepared for surgery. After weighing, each individual was premedicated. On the day of surgery, each animal in the group was sedated by intramuscular injection (Domitor-Orion Corporation, Fin-land) of medetomidine (0.5 mg / kg) and Butomidor, Richter Pharma, Austria) butorphanol (0.2 mg / kg) depending on their weight. Then, after about 15 minutes, a mask was put on in order to administer inhalation anesthesia (isofluorane).
The period of anesthesia of each individual lasted about 30 minutes. After the rabbit was immobilized, the skin was shaved, disinfected with alcohol and iodine. The material for implantation was prepared according to the recommendations.
The skin incision was made parallel, in the intercostal area, in the middle of the latissimus dorsi muscle length, 3 cm above the dorsal line. Subcutaneous tissue and fascia were dissected in the same line and the prepared material was placed (cylindrical shape height 1 cm, diameter 1,5 cm). Two materials were implanted into the one organism (one on the left one on the right side). Experimental samples were chitosan modified by gallic acid (CTS_GA), ferulic acid (CTS_FA) and tannic acid (CTS_TA). Control was prepared by implantation of chitosan scaffold without addition of phenolic acid (CTS). The implantation site was closed with a mattress suture using Dexon 3 − 0.
After the operation, all the rabbits could move freely in the cages without additional dressings in the area operated on. In order to minimize the risk of infection and reduce postoperative discomfort, an antibiotic and an anti-inflammatory drug (gentamicin 5 mg / kg and meloxicam 0.4 mg / kg) were administered for 5 days after the procedure.
In the postoperative period, a mild swelling was observed around the skin suture in most rabbits. After two weeks, all the operated animals were in good general condition. Three months after surgery, all rabbits were sacrificed. First, animals were anesthetized intramuscularly and sedated by intramuscular injection of medetomidine (0.5 mg / kg) and butorphanol (0.2 mg / kg) depending on their weight.
The rabbits were then sacrificed by barbiturate injection. Tissue fragments with a margin (3cm x 3cm x 3cm) were taken from the implantation site along with the implanted material and placed in a buffered paraformaldehyde solution at pH 7.4. All the samples were placed in appropriate transporters and accurately described according to the implanted material.
All the animal surgical procedures were carried and approved by the Local Ethical Committee for Experiments on Animals in Lublin (Agreement no. 104/2017).
Histological Assessment
Tissue samples were immediately fixed in 10% buffered formalin, processed routinely for histopathology using paraffin method, cut at 5µm and stained with Mayer’s haematoxylin and eosin. Samples were evaluated in a blind fashion by an experienced pathologist (IOD). Microphotographs were prepared using Olympus BX43 microscope (Tokyo, Japan), equipped with Olympus SC 180 camera (Hamburg, Germany) and cellSens software (Olympus).