Effects Of Mechanical Compression On Human Corneal Fibroblasts

Objective: To isolate,culture,and identify human corneal fibroblasts derived from Small incision lenticule extraction(SMILE).To explore the effects of mechanical compression on the cellular morphology,cell proliferation and apoptosis,and expression of factors involved in corneal matrix synthesis and degeneration of human corneal fibroblasts.Methods: Human corneal fibroblasts were isolated and cultured after collagenase type I digestion from corneal stromal lenticule obtained from SMILE surgery.The cellular morphology was observed under an inverted microscope.Immunofluorescent staining of Vimentin was used to identify the fibroblasts.An in vitro cell mechanical compression model was established to give corneal fibroblasts pressure(Agar+0g,Agar+1g,Agar+2g,Agar+4g,Agar+8g,Agar+16g,Agar+24g,Agar +32g),and the stimulation was given for 6h,12 h,and 48 h.Morphological changes were observed under an inverted microscope.BrdU staining was used to detect the proliferation,and Annexin V/PI staining was used to determine the apoptosis of human corneal fibroblasts under mechanical compression.qRT-PCR was used to investigate the mRNA expression of factors involved in corneal matrix synthesis and degeneration,including matrix metalloproteinases(MMPs,including MMP1,MMP9),tissue inhibitor of matrix metalloproteinases(TIMPs,including TIMP1,TIMP2,TIMP3),and corneal structural proteins(including Col1a1,Vimentin,Lumican).Results: 1.Human corneal fibroblasts can be isolated and cultured from corneal stromal lenticule obtained from SMILE surgery by collagenase digestion.The cells were long spindle-shaped or triangular-shaped,which was consistent with the characteristics of corneal fibroblasts.The cells were identified as pure corneal fibroblasts with Vimentin-positive.2.We successfully established a mechanical compression stimulation model.Human corneal fibroblasts maintained their original morphological characteristics under mechanical compression with relative low-pressure level and short stimulation time.However,with the increase of pressure level and stimulation time,corneal fibroblasts showed significant morphological alterations in Agar+32g group stimulated for 6h,Agar+16g group stimulated for 48 h,and Agar+24g group stimulated for 48 h.Corneal fibroblasts under excessive high-pressure level with long stimulation time became larger in cell size,and the cytoplasmic processes became shorter and thicker.The nucleus became larger,and several cells showed nuclear pyknosis.3.There was no significant difference in the ratio of BrdU-positive cells between control group and Agar+0g group.But the proportion of BrdU-positive cells in the Agar+8g group significantly decreased when compared to control,indicating that mechanical compression inhibited cell proliferation.In addition,Annexin V/PI staining showed that the percentage of Annexin V-positive cells and PI-positive cells in Agar+8g group increased after 8h of mechanical compression in comparison with control,implicating that mechanical compression promoted cell apoptosis.4.Significant elevation of MMP1 and MMP9,moderate increase of TIMP1 and Lumican,and moderate reduction of TIMP3 were observed 6h after mechanical compression in almost all compressed groups.When corneal fibroblasts were stimulated for 24 h,MMP1 elevated while Col1a1 reduced with the increase of pressure,and MMP9 significantly elevated in most compressed groups.When corneal fibroblasts were treated with mechanical compression for 48 h,MMP1 significantly up-regulated,and genes encoding corneal structural proteins including Col1a1,Vimentin,and Lumican significantly down-regulated after mechanical compression.Conclusion: 1.It is an effective way to obtain sufficient and purified primary human corneal fibroblasts by digesting corneal stromal lenticule tissue from SMILE surgery with collagenase.2.Human corneal fibroblasts will lose their original morphological characteristics when applied with excessive mechanical compression.3.Mechanical compression inhibits proliferation and promotes apoptosis of human corneal fibroblasts.4.Mechanical compression can promote the expression of MMPs such as MMP1 and MMP9 in the early stage of compression,and inhibit the expression of corneal structural proteins such as Col1a1,Lumican,and Vimentin in the late stage of compression,which may participate in determining the structure of human corneal stroma.