PMID- 33238685 OWN - NLM STAT- MEDLINE DCOM- 20201221 LR - 20201221 IS - 1009-2587 (Print) IS - 1009-2587 (Linking) VI - 36 IP - 11 DP - 2020 Nov 20 TI - [Research on feasibility of in vitro inflammatory wound microenvironment simulated by using inflammatory wound tissue homogenate of mice]. PG - 1024-1034 LID - 10.3760/cma.j.cn501120-20200720-00351 [doi] AB - Objective: To investigate the feasibility of in vitro inflammatory wound microenvironment simulated by using inflammatory wound tissue homogenate of mice. Methods: (1) Ten eight-week-old C57BL/6 male mice were collected and full-thickness skin tissue with diameter of 1.0 cm on both sides of the midline of the back was taken with a perforator to make the normal skin tissue homogenate supernatant. At 48 h after the full-thickness skin defect wound was established, the wound tissue within 2 mm from the wound edge was taken to make inflammatory wound tissue homogenate supernatant. Two kinds of tissue homogenate supernatant were taken to adjust the total protein concentration to 1 mg/mL, and the tumor necrosis factor alpha (TNF-alpha) content was detected by enzyme-linked immunosorbent assay. The number of sample was 6. (2) The primary passage of human umbilical cord mesenchymal stem cells (hUCMSCs) were collected and cultured to the 3rd passage with the normal exosomes being extracted from the hUCMSCs after cultured for 48 h. Another batch of hUCMSCs in the 3rd passage was collected and stimulated with inflammatory wound tissue homogenate supernatant of 30, 50, and 100 mug/mL total protein and normal skin tissue homogenate supernatant of 30, 50, and 100 mug/mL total protein, respectively. After cultured for 48 h, the exosomes stimulated with normal protein of 30, 50, and 100 mug/mL and exosomes stimulated with inflammatory protein of 30, 50, and 100 mug/mL were extracted. Normal exosomes, exosomes stimulated with 30 mug/mL normal protein, and exosomes stimulated with 30 mug/mL inflammatory protein were collected, the morphology was observed by transmission electron microscope, the particle size was detected by nanoparticle tracking analyzer, and the expressions of CD9 and CD63 were detected by Western blotting. (3) Twenty one-day-old C57BL/6 mice were taken to isolate the primary passage of fibroblasts (Fbs) and the 3rd passage of Fbs, whose morphology was observed under the inverted phase contrast microscope. The Fbs of 3rd passage were collected to observe the expression of vimentin by cell crawling method combined with immunofluorescence method at culture hour (CH) 2. (4) The Fbs of 3rd passage were divided into control group, normal exosome group, 30, 50, 100 mug/mL normal protein stimulating exosome group, and 30, 50, 100 mug/mL inflammatory protein stimulating exosome group according to the random number table, with 4 wells in each group. Cells in control group received no treatment, and cells in the other 7 groups were respectively added with normal exosomes, exosomes stimulated with normal protein of 30, 50, and 100 mug/mL, and exosomes stimulated with inflammatory protein of 30, 50, and 100 mug/mL prepared in experiment (2). The final mass concentration of exosomes was adjusted to 10 mug/mL. The cell viability was detected by cell count kit 8 at CH 48. (5) Two batches of Fbs in the 3rd passage were divided and treated as those in experiment (4), with 4 wells in each group, and the final mass concentration of exosomes was adjusted to 1 and 10 mug/mL, respectively. The cell mobility was detected by cell scratch test at CH 6, 12, and 24. (6) Two batches of the Fbs of 3rd passage were collected, divided, and treated as those in experiment (4) except with no control group, with 3 wells in each group, and the final mass concentration of exosomes was respectively adjusted to 1 and 10 mug/mL. The mRNA expression levels of transforming growth factor beta(1) (TGF-beta(1)), TGF-beta(3), and alpha smooth muscle actin (alpha-SMA) were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction at CH 48. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and Bonferroni method. Results: (1) The content of TNF-alpha in inflammatory wound tissue homogenate supernatant of mice was (116+/-3) pg/mL, significantly higher than (97+/-5) pg/mL in normal skin tissue homogenate supernatant at post injury hour 48 (t=3.306, P<0.05). (2) Normal exosomes, exosomes stimulated with 30 mug/mL normal protein, and exosomes stimulated with 30 mug/mL inflammatory protein of hUCMSCs showed the typical saucer-like shape. The particle sizes of the three exosomes of hUCMSCs were 30-150 nm, which were all within the normal particle size range of exosome. Three exosomes of hUCMSCs positively expressed CD9 and CD63. (3) The primary passage of cells were clearly defined and showed protruding spindle shape, irregular polygon shape, or slender strip shape. The morphology of the 3rd and the primary passage of cells is similar. At CH 2, vimentin in cells was positively expressed, and the cells were identified as Fbs. (4) At CH 48, the cell viability was (137.4+/-2.8)% in 30 mug/mL inflammatory protein stimulating exosome group, obviously higher than 100%, (107.5+/-2.4)%, (113.3+/-3.2)%, (104.0+/-2.0)%, and (101.9+/-1.5)% in control group, normal exosome group, 30 mug/mL normal protein stimulating exosome group, and 50 and 100 mug/mL inflammatory protein stimulating exosome groups, respectively (P<0.01), and cell viability in 30 mug/mL normal protein stimulating exosome group was obviously higher than that in control group, normal exosome group, and 50 and 100 mug/mL normal protein stimulating exosome groups [(103.4+/-2.2)% and (102.5+/-1.4)%], respectively (P<0.01). (5) At CH 6, 12, and 24, the mobility rate of cells in 30 mug/mL inflammatory protein stimulating exosome group was significantly higher than that in control group, normal exosome group, 30 mug/mL normal protein stimulating exosome group, and 50 and 100 mug/mL inflammatory protein stimulating exosome groups, respectively, when the final mass concentrations of exosome was 1 mug/mL (P<0.05) . At CH 12, the mobility rate of cells in 30 mug/mL normal protein stimulating exosome group was obviously higher than that in control group, normal exosome group, and 50 and 100 mug/mL normal protein stimulating exosome groups, respectively, when the final mass concentration of exosome was 1 mug/mL (P<0.05). At CH 6, the mobility rate of cells in 30 mug/mL inflammatory protein stimulating exosome group was significantly higher than that in control group and normal exosome group (P<0.05), and the mobility rate of cells in 30 mug/mL normal protein stimulating exosome group was significantly higher than that in 50 and 100 mug/mL normal protein stimulating exosome groups, respectively, when the final mass concentration of exosome was 10 mug/mL (P<0.05). At CH 12 and 24, the mobility rate of cells in 30 mug/mL inflammatory protein stimulating exosome group was significantly higher than that in control group, normal exosome group, and 50 and 100 mug/mL inflammatory protein stimulating exosome groups (P<0.05), and the mobility rate of cells in 30 mug/mL normal protein stimulating exosome group was significantly higher than that in control group, normal exosome group, and 50 and 100 mug/mL normal protein stimulating exosome groups, respectively, when the final mass concentration of exosome was 10 mug/mL (P<0.05). (6) There were no statistically significant differences in mRNA expression levels of TGF-beta(1), TGF-beta(3), and alpha-SMA of cells among the 7 groups at CH 48 when the final mass concentration of exosome was 1 mug/mL (F=1.123, 1.537, 1.653, P>0.05). There were no statistically significant differences in mRNA expression levels of TGF-beta(1) and alpha-SMA of cells among the 7 groups at CH 48 when the final mass concentration of exosome was 10 mug/mL (F=1.487, 1.308, P>0.05), and mRNA expression level of TGF-beta(3) of cells in 50 mug/mL inflammatory protein stimulating exosome group at CH 48 was significantly higher than that in normal exosome group, 50 mug/mL normal protein stimulating exosome group, and 30 and 100 mug/mL inflammatory protein stimulating exosome groups when the final mass concentration of exosome was 10 mug/mL (P<0.05). Conclusions: The pretreatment with inflammatory wound tissue homogenate supernatant of mice has no significant effect on the total protein of hUCMSCs exosomes. The hUCMSCs exosomes stimulated by low concentration inflammatory wound tissue homogenate supernatant can significantly promote the proliferation and migration ability of Fbs. The content of inflammatory mediators in the wound tissue homogenate supernatant during the inflammatory phase is extremely low, which may be the reason that the anti-inflammation and tissue repair paracrine effects of mesenchymal stem cell cannot be effectively started. FAU - Hao, Y AU - Hao Y AD - Department of Burns and Plastic Surgery, the Affiliated Hospital of Zunyi Medical University, Zunyi 563003, China. FAU - Yang, Q X AU - Yang QX AD - Department of Burns and Plastic Surgery, the Affiliated Hospital of Zunyi Medical University, Zunyi 563003, China. FAU - Wang, Q AU - Wang Q AD - Department of Burns and Plastic Surgery, the Affiliated Hospital of Zunyi Medical University, Zunyi 563003, China. FAU - Xu, G C AU - Xu GC AD - Department of Burns and Plastic Surgery, the Affiliated Hospital of Zunyi Medical University, Zunyi 563003, China. FAU - Qi, F AU - Qi F AD - Department of Burns and Plastic Surgery, the Affiliated Hospital of Zunyi Medical University, Zunyi 563003, China. FAU - Deng, C L AU - Deng CL AD - Department of Burns and Plastic Surgery, the Affiliated Hospital of Zunyi Medical University, Zunyi 563003, China. FAU - Wei, Z R AU - Wei ZR AD - Department of Burns and Plastic Surgery, the Affiliated Hospital of Zunyi Medical University, Zunyi 563003, China. FAU - Wang, D L AU - Wang DL AD - Department of Burns and Plastic Surgery, the Affiliated Hospital of Zunyi Medical University, Zunyi 563003, China. LA - chi GR - 81871570, 82072195/General Program of National Natural Science Foundation of China/ GR - No. 20162910, 20204Y148/Science and Technology Plan of Guizhou Province/ GR - No. 201507/Collaborative Innovation Center of 2011 for Tissue Injury Repair and Regenerative Medicine of Guizhou Province/ PT - Journal Article PL - China TA - Zhonghua Shao Shang Za Zhi JT - Zhonghua shao shang za zhi = Zhonghua shaoshang zazhi = Chinese journal of burns JID - 100959418 SB - IM MH - Animals MH - Cell Movement MH - Feasibility Studies MH - Fibroblasts MH - *Mesenchymal Stem Cells MH - Mice MH - Mice, Inbred C57BL OTO - NOTNLM OT - Exosomes OT - Fibroblasts OT - Human umbilical cord mesenchymal stem cells OT - Inflammation OT - Microenvironment OT - Tissue homogenate EDAT- 2020/11/27 06:00 MHDA- 2020/12/22 06:00 CRDT- 2020/11/26 03:23 PHST- 2020/11/26 03:23 [entrez] PHST- 2020/11/27 06:00 [pubmed] PHST- 2020/12/22 06:00 [medline] AID - 10.3760/cma.j.cn501120-20200720-00351 [doi] PST - ppublish SO - Zhonghua Shao Shang Za Zhi. 2020 Nov 20;36(11):1024-1034. doi: 10.3760/cma.j.cn501120-20200720-00351.