To establish in vitro 3D model of cell culture and pressure with Flexcell-5000C pressure system and rat tail type I collagen to explore the in vitro effect of pressure on hypertrophic scar fibroblasts (HSFb) at cellular level.

The primary HSFbs were isolated and cultured by collagenase digestion. A 3D culture model of HSFb was constructed in vitro with Flexcell-5000C pressure system and rat tail type I collagen. All the HSFbs were randomly divided into control group (no pressure) and 10 mmHg , 20 mmHg and 30 mmHg pressure groups. The cytomorphosis of HSFbs in different pressure was monitored by immunofluorescence. The cellular proliferation index of HSFbs was examined by ki67/EdU immunofluorescence staining of chromosome. The HSFbs apoptosis was detected by Annexin-V-PI double flow cytometry. The cell cycle was detected by PI single staining flow cytometry and cell migration rate, by scratch assay. Data were processed with one-way analysis of variance and the LSD-t test.

Examined by confocal microscope, the morhpological changes happened in HSFbs under mechanical pressure. The HSFb phenotype was gradually transferred to normal fibroblast (FB) along with the increase of the pressure, especially under 30 mmHg pressure. It was indicated by Ki-67/Edu immunofluorescence staining that the proliferation capacity of HSFbs was inversely proportional to the pressure value. The proliferation value of HSFbs in 20 mmHg and 30 mmHg groups was evidently lower than that in control group, the difference was statistically significant (F=10.61, P=0.0037). But there was no significant difference of the proliferation value among all the other groups (P>0.05). It was indicated by Annexin-V-PI double flow cytometry that the early apoptosis rate in 30 mmHg group was much higher than that in the control, 10 mmHg and 20 mmHg groups (F=103.8, P<0.0001). And there was no significant difference of apoptosis between 10 mmHg, 20 mmHg and control groups(P> 0.05). It was exhibited by PI single staining flow cytometry that the HSFb percentage in 30 mmHg group increased in G0/G1 phases(F=21.58, P=0.0003) and decreased in S and G2/M phases significantly(F=93.89、54.11, P<0.001、=0.0066), when compared with that in control 10 mmHg and 20 mmHg groups. Furthermore, the HSFb percentage in G0/G1 phases in the 10 mmHg and 20 mmHg groups was much higher than that in control group, the difference was statistically significant (P<0.05), but the percentage in S and G2/M phases decreased obviously in 30 mmHg group (P<0.05). Nevertheless, there was no significant difference in terms of cell cycle between 20 mmHg and 10 mmHg groups (P>0.05). It was examined by scratch assay that the migration ability of HSFB in 30 mmHg pressure group was significantly decreased (t=10.66, P=0.004).

It was indicated that the in vitro 3D model of cell culture and pressure with Flexcell-5000C pressure system and rat tail type I collagen established in this study exhibited stable and handy with real time controllable and adjustable pressure value. With this model, it was successfully verified that pressure could inhibit the proliferation and migration but accelerate the apoptosis and transdifferentiation to Fb of HSFbs, in which 30 mmHg was testified to be the best pressure value. The study raises a new scientific idea and provide in vitro model for the pressure management of hypertrophic scar.