Expression of GFP-tag and observation of green color as confirmation for transfection of expression plasmids in human Dermal fibroblast cells
The transfection of HDF cells with pLenti-III plasmids was successful with a 20–25% transaction rate as estimated after 48 hours of transfection. The confirmation of the transfection was done using a fluorescence microscope, and the results were presented in Fig. 2.
The expression level of miR-192 was analyzed by flow cytometry using the FL1 channel compared to fresh and untransfected cells, the results of this analysis were presented in Fig. 3 of the study.
Expression of miR-192 in transfected fibroblast cells increased COL1A2 gene expression
The results of this analysis showed a significant increase (3-fold) in miR-192 expression in the fibroblast cells transfected with the miR-192 expressing plasmid compared to the cells transfected with the backbone expressing plasmid after 48 hours, as shown in Fig. 4, this confirmed the successful transfection of the cells and the overexpression of miR-192 in the transfected cells.
The study used RT-qPCR to test if miR-192 increased in fibroblast cells would change COL1A2 expression, Results showed a 15.77-fold increase in COL1A2 expression in transfected cells compared to the control group after 48 hours (Fig. 5(, suggests miR-192 positively regulates COL1A2 expression and a potential mechanism for its effects on wound healing.
Simultaneous treatment with HDF cells expressing miR-192 and PRP caused more wound closure than other groups.
On the 3rd day, a significant difference was seen among the group receiving HDFs expressing pLenti-III-Backbone-GFP and the group treated with HDFs expressing pLenti-III-miR-192-GFP (P < 0 /0001) .There was a significant difference between the group that received PRP and HDFs expressing pLenti-III-miR-192-GFP and other treatment groups P < 0.0001 and more wound closure have occurred (Fig. 6).
On the seventh day after wound formation, the wounds were examined macroscopically, and measurements were taken and recorded with a digital calliper. On the seventh day, the wound healing process accelerated in all intervention groups compared to the third day, and the difference between the groups became more obvious. According to the order of the groups in Fig. 7, the average percentage of wound closure in various intervention groups versus the control groups is as follows: 68/33, 60/67, 66/83, 83/00, 91/53, and 89/03 percent.
A significant difference of P = 0.0001 was seen in the healthy control group and the miR-192-expressing, PRP-group, and combined treatment groups, and these groups healed wounds quicker than the healthy control group. On the seventh day, the diabetic control group that did not receive treatment experienced a delay in the healing of the wound compared to the other groups. The difference between the rates of wound closure in the treated groups compared to the diabetic control group was almost completely different in the treated groups. Most of the wound area is closed, especially in the fifth and sixth groups, where it ranges from 80 to 90%, with diabetic wounds being close to 60%. In comparison to the group that only received fibroblast cells expressing pLenti-III-miR-192-GFP, more wounds closed in the combination therapy group (P = 0.0001). (Fig. 7)
On the fourteenth day, there was a significant difference between the HDF groups expressing pLenti-III-Backbone-GFP and pLenti-III-miR-192-GFP plasmids (P < 0.0001), and more wound closure was observed in the HDF group expressing pLenti-III-miR-192-GFP. Between the PRP-treated group and the HDF-expressed group, there was a smaller difference (P < 0.05). The group that received the combination therapy with the groups treated with PRP and fibroblasts experiencing pLenti-III-miR-192-GFP alone is significant, with P < 0.0001 and NS (Not significant) respectively.
On the 21st day after wound formation, healing occurred in almost all intervention groups, but the diabetic group without treatment has the lowest average percentage of wound closure (88.48%) compared to other groups, the group that simultaneously received PRP and fibroblast cells expressing pLenti-III-miR-192-GFP showed significant differences with each group that received the mentioned treatments alone, the group that was treated with combined treatment, the rate of wound closure and scars left from healing was lower than in other groups. (Fig. 8). The macroscopic views of the wound on different days are shown in Fig. 9.
The number of fibroblast cells in the group that simultaneously received HDF cells expressing miR-192 and PRP was higher than in the other groups.
On the 14th and 21st days following the development of a wound, the average number of fibroblast cells was counted in the dermis of the wound area in various intervention groups. Figure 10 shows that the group that received the combined treatment had more fibroblast cells than the other groups. The fact that there was a noticeable difference between this group and the groups which received each treatment separately could mean that the injected fibroblast cells in the wound area were functional and alive.
The density of collagen fibers in the group treated with PRP and HDF cells expressing miR-192 increased compared to other groups
The density of collagen fibers in the group treated with PRP and HDF cells expressing miR-192 increased compared to other groups the group that got the combination therapy and HDF expressing pLenti-III-miR-192-GFP had a higher collagen fiber density on day 14 than the other groups, and there was a significant difference between these two groups and the PRP-treated group. A notable difference was observed between the groups that got HDF containing pLenti-III-miR-192-GFP plasmid and the backbone group on the 21st day following injury, additionally compared to each of the treatment groups separately, the combined treatment group's collagen fiber density increased, however, this rise was not statistically significant (Fig. 11).
Figure 12 shows Masson-trichrome staining of the wound area in different groups on the 14th day after wound induction.
Combined treatment with PRP and HDF cells expressing miR-192 increased the area of granulation tissue and wound healing.
On the seventh day, the group treated at the same time with PRP and HDF expressing pLenti-III-miR-192-GFP, the average area of the granulation tissue is 502188 µm square and more than other groups, and there is a critical difference between this group and the two groups treated with PRP and HDF containing pLenti-III-miR-192-GFP expression plasmids alone was seen P < 0.0001.
On the fourteenth day, a noteworthy distinction was observed between the group that received the combined treatment and the group that was treated with a PRP P < 0.05. But there was no difference between the combined group and the group that had fibroblasts containing pLenti-III-miR-192-GFP. There was no noteworthy difference between the PRP group and the group treated with HDF expressing pLenti-III-miR-192-GFP. But in all the groups that received treatment, compared to the healthy and diabetic control groups, there was a critical distinction, and the area of the granulation tissue was greater within the intervention groups than within the control groups. (Figure 13). The area of granulation tissue can be seen in hematoxylin-eosin staining in Figure 14.