Animals and study design

The study was conducted on 35 Sprague–Dawley albino rats. Five male rats aged 3 weeks (27–32 g) were used for bone marrow isolation and thirty female rats weighing 150–200 g 6–8 weeks of age were used in the experimental groups. Rats were allowed to acclimate for 2 weeks before the experiment and were housed under a 14–10 h light–dark cycle with food and water provided ad libitum. Experiments were conducted in accordance with the approved guidelines set by the Research Ethics Committee of Alexandria Faculty of Medicine, Egypt.

Stem cell processing was carried out in the Center of Excellence for Research in Regenerative Medicine and its Application (CERRMA), Alexandria Faculty of Medicine. Ethical approval was attained from the Medical Ethics Committee of Alexandria Faculty of Medicine (IRB NO: 00012098-FWA NO: 00018699).

The 30 female rats were divided randomly into 2 groups;

Control Group, n = 10, which was further subdivided into a negative control group (CG) and a positive control group, in the latter lung fibrosis was induced for optimization of model and named fibrotic group (FG). These groups were given an intratracheal injection of saline or a single dose of bleomycin (5 mg/kg body weight, in 0.4 ml of saline) respectively. Vial of bleomycin powder was dissolved in 5 ml saline. Animals were held in `upright position by an assistant and the neck was pulled backward. A syringe filled with BLM was fitted to an orogastric tube, and then was pushed gently against the soft palate to reach the trachea. Induction of lung fibrosis was assessed 28 days after bleomycin injection [17].

Experimental Group (where lung fibrosis was induced) EG n = 20, then it was subdivided into; Bone marrow-derived mesenchymal stem cells treated group (BM-MSCsG) where rats were injected with a single dose of passage 3 (P3) BM-MSCs in complete media, and cell free media treated group (CFG) where rats were injected once with cell free complete media, intravenously 28 days after induction of fibrosis. Histological assessment was done after another 28 days from treatment and was further supported by physiological assessment of lung functions.

The bleomycin used in the current study was commercially purchased as Bleocel 15 IU Injection: bleomycin sulfate powder for solution for injection/infusion manufactured by celon Labs.