Effects of hypoxia-pretreated rat adipose-derived mesenchymal stem cells conditioned medium on wound healing of rats with full-thickness defects

Objective To investigate the effects of hypoxia-pretreated rat adipose-derived mesenchymal stem cells (ADSCs) conditioned medium on wound healing of rats with full-thickness defects. Methods (1) A 6-week-old male Sprague-Dawley rat was sacrificed by cervical dislocation, the bilateral inguinal adipose tissue was collected, the third generation ADSCs were isolated by collagenase digestion method, and the cells morphology was observed. The cells were harvested and divided into adipogenic induction group and osteogenic induction group according to the random number table (the same grouping method below), with 6 wells in each group. The cells in adipogenic induction group were cultured for 14 days to observe adipogenesis, and cells in osteogenic induction group were cultured for 28 days to observe osteogenesis. (2) The third generation ADSCs were collected and divided into normoxic group and hypoxic group. Cells in normoxic group was incubated in normal oxygen incubator with oxygen volume fraction of 21%, and cells in hypoxic group was incubated in low oxygen incubator with oxygen volume fraction of 2% respectively, with 3 samples in each group for each time point. Three samples in normoxic group on 3 h of culture and in hypoxic group on 3, 6, 12, 24, and 48 h of culture were collected for detecting the following indexes. The mRNA expressions of hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and peroxisome proliferator-activated receptor γ (PPAR-γ) were detected by real time fluorescent quantitative reverse transcription polymerase chain reaction. The cell culture supernatant in the two groups was collected, centrifuged, and filtered to obtain normoxic conditioned medium (normo-CM ) and hypoxic conditioned medium (hypo-CM). Enzyme linked immunosorbent assay was used to detect content of VEGF, transforming growth factor β (TGF-β), epidermal growth factor (EGF), and insulin-like growth factor (IGF) in conditioned medium. (3) Twenty-seven male Sprague-Dawley rats aged 6-8 weeks were collected and divided into phosphate buffer solution (PBS) group, normo-CM group, and hypo-CM group, with 9 rats in each group. A circular full-thickness skin defect wound with diameter of 1 cm was made on the back of each rat, and the wounds of rats in PBS, normo-CM, and hypo-CM groups were respectively dropped with 50 μL PBS, normo-CM, and hypo-CM. On post injury day (PID) 0, 3, 5, 7, 9, and 11, the gross condition of wound was observed, wound area was measured, and the non-healing rate of wound was calculated. The wound tissue was collected for hematoxylin eosin staining to observe inflammatory reaction of wound on PID 3, 9, and 11 and re-epithelialization of wound on PID 9. Masson staining was used to observe the collagen deposition and analyze collagen volume fraction of wound on PID 11. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, t test, and Bonferroni correction. Results (1) The isolated cells showed a fusiform, in adherent growth and close arrangement when in low fusion degree. On 14 d of culture, the red lipid droplets stained with oil red O were observed in cells in adipogenic induction group, and on 28 d of culture, the red nodules stained with alizarin red S were observed in cells in osteogenic induction group. The cells were identified as ADSCs. (2) Compared with that in normoxic group, the mRNA expression of HIF-1α was significantly increased at 12 and 24 h of culture (t=5.43, 5.11, P<0.05), the mRNA expression of VEGF was significantly increased at 6 and 12 h of culture (t=3.29, 2.33, P<0.05 or P<0.01), the mRNA expression of bFGF was significantly increased at 12 h of culture (t=12.59, P<0.01) and significantly reduced at 48 h of culture (t=9.34, P<0.01), and the mRNA expression of PPAR-γ was significantly reduced at 3, 12, and 24 h of culture in hypoxic group (t=5.14, 6.56, 4.97, P<0.05). (3) Compared with that in normoxic group, the VEGF content was significantly increased at 3, 6, 12, 24, and 48 h of culture (t=5.74, 12.37, 14.80, 15.70, 34.63, P<0.05 or P<0.01), and the IGF content was significantly increased at 6, 12, 24, and 48 h of culture (t=5.65, 8.06, 20.12, 22.99, P<0.05 or P<0.01), and the content of TGF-β and EGF showed no obvious change at 3, 6, 12, 24, and 48 h of culture in hypoxic group. (4) From PID 0 to 11, the wound of rats in the three groups shrank to varying degrees, with no obvious infection or exudate. On PID 11, the wound area of rats in PBS group was still large, which was larger than that in normo-CM group, and the wound area of rats in hypo-CM group was basically healed. On PID 0, 3, and 5, the non-healing rates of wound of rats in the three groups were similar. On PID 7, the non-healing rates of wound of rats in normo-CM and hypo-CM groups were significantly lower than that in PBS group (t=10.26, 16.03, P<0.05). On PID 9, the non-healing rate of wound of rats in hypo-CM group was significantly lower than that of PBS group and normo-CM group, respectively (t=17.25, 6.89, P<0.05 or P<0.01), and the non-healing rate of wound of rats in normo-CM group was significantly lower than that in PBS group (t=8.81, P<0.05). On PID 11, the non-healing rate of wound of rats in hypo-CM group was (2.4±1.5)%, which was significantly lower than (20.0±5.0)% in PBS group and (7.7±1.7)% in normo-CM group (t= 30.15, 84.80, P<0.05). (5) On PID 3, the infiltration of inflammatory cells in the wound of rats in hypo-CM group was obviously more than those in the other two groups. On PID 9, the infiltration of inflammatory cells in the wound of rats in hypo-CM and normo-CM groups was obviously less than that in PBS group. On PID 11, the infiltration of inflammatory cells in the wound of rats in hypo-CM group was obviously less than those in PBS and normo-CM groups. On PID 9, the length of " epidermal migration tongue" on the wound of rats in hypo-CM group was longer than those of the other two groups, and the epidermis thickness was close to normal skin. On PID 11, compared with those in PBS and normo-CM groups, a large number of collagen deposits with dense structure, neat arrangement, and higher maturity were seen in the wound of rats in hypo-CM group. The wound collagen volume fraction of rats in PBS group was (22.90±1.25)%, which was significantly lower than (31.96±0.14)% in normo-CM group and (56.10±1.50)% in hypo-CM group (t=12.48, 29.43, P<0.05), and the wound collagen volume fraction of rats in normo-CM group was significantly lower than that in hypo-CM group (t=27.73, P<0.05). Conclusions Hypoxia-pretreated can significantly enhance paracrine effect of rat ADSCs. Hypoxia-pretreated rat ADSC conditioned medium can accelerate the healing of full-thickness skin defect wound in rats by regulating inflammatory cell infiltration, promoting re-epithelialization and collagen deposition in the wound.