All the techniques were accepted by the Medical Ethics
Commission of Lorestan university medical of sciences, Khoramabad, Iran
(protocol no: LUMS.REC.2016.147-23/8/2016).BMMSCs were isolated from rats?long
bones, and expanded. The expanded MSCs were used for immunophenotypic analysis.
The cells were
characterized with regard to a set of markers characteristic for BMMSCs
including CD90, CD45, CD105 and CD34. Immunization of
cells with fluorescent stain were performed. 6 × 107
BMMSCs were injected to each 12 points of each flap after surgery. CEE were
isolated from 9-day- old embryos. A
30×80-mm RSF were made in dorsum of forty rats. The rats were divided into four groups as follow.
Group one did not receive any treatment and was served as control, group 2 received
BMMSCs, group 3 received CEE+ BMMSCs, and
group 4 received CEE. Seven days after surgery, survived part of
each flap were measured, and a sample from transition line were prepared for
histological study and blood vessel sections, MCs number, and number of MCs degranulation were examined.
MSC Isolation, Culture, and Labelling
A group of adult healthy rats were euthanized and BMMSCs
were harvested from the bilateral tibias and femurs. Briefly, rats were
euthanized, then the long bones were extracted aseptically and the bone marrow
flushed with Dulbecco’s Modified Eagle Medium (DMEM) from the medullary cavity
of the bones. The procedure has been described completely before (16,17).
Briefly the BMMSCs were labelled with fluorescent molecule DiI (1?-Dioctadecyl-3,3,3?,3?-Tetramethylindocarbocyanine
Perchlorate Molecular Probes) with a ratio of 1:100 and suspended in 0.5 ml
DMEM for transplantation. 6 × 107 BMMSCs were homogenized within 0.5 ml DMEM and
were used for each injection.
Viability of BM MSCs
Trypan blue staining was used to discriminate between
viable and non?viable cells as described completely before (18). Briefly
the diluted cell samples in normal saline were prepared. Trypan blue was mixed with
cell suspension and following proper pipetting, 10 ?l of solution was loaded on
a hemocytometer slide, and were counted under a microscope in all four squares
of the hemocytometer chambers, and the average number of cells per square (109
per ml) was determined.
Preparation of Chicken Embryo Extract
CEE was isolated from 9?day?old embryos (Spafas, Lancaster, PA, USA) under sterile conditions according to the method that
were described completely before (18, 17). Briefly, the embryos were washed
3 times in Phosphate-Buffered Saline (PBS) before
homogenization through a 50-cc syringe. The homogenate was then passed through
a 22-gauge needle. The homogenate was diluted in DMEM and pelleted by
centrifugation. The supernatant (CEE) was extracted and saved. The CEE was
passed through a 0.45- µm filter for sterilization, and the protein
concentration was determined using the Bradford method (18,17). 0.5 ml of CEE
were used for each injection.