| Objective: Reducing formation of scar and improving the healing quality of skin incision has become a research hotspot in the modern trauma repair field. The healing of skin incision is a complex and orderly biological process, in which growth factors play an important role in. Autologous Concentrated Growth Fators(CGF), which contains a variety of growth factors, plays an important role in tissue trauma repair. Histopathology method and morphologic measurement are applied to investigate the effect of CGF liquid on the healing of skin incision. The study aim at providing a guide for the clinical application by observing the effect of CGF liquid on skin incision of New Zealand white rabbits. 1 Experimental animals and groups Twelve healthy male New Zealand rabbits with age of twelve months and weights from 2.5 to 2.8 kilogram were selected for the study. There were six incisions on the back of each rabbit with self-contrast and there were 72 incisions in the experiment. The animal samples were divided into three groups: group A(injection of CGF for two times), group B(injection of CGF for one time), group C(injection of nothing). 2 The preparation of CGF liquid Nine milliliters venous blood from the ear middle artery of each rabbit was collected into 9 ml sterile Vacuette tubes to prepare CGF liquid. After differential centrifugation, 4 ml CGF liquid was absorbed from the CGF layer with syringe for use. 3 The procedure of operation Under general anesthesia, 6 skin incisions were made on back of each rabbit. The length of the incisions was 2.0 cm and the incisions were parallelto the spine. The depth of the incisions included skin, subcutaneous tissue, and muscle. Before suture, 1ml CGF liquid was injected into the edge of incision of group A and group B and anything was not injected into incisions of group C. After one week, 1 ml CGF liquid was injected into the edge of incision of group A again. Anti-infective therapy was given to the rabbits after surgery. The stitches was taken away from incisions after one week. 4 Collection of samples On the 1st, 2nd, 3rd, 4th weeks after the surgery, 3 rabbits were sacrificed at each time point and specimens were collected according to different groups. Every specimen included the full-thickness skin with the area 2.0 x 1.0 cm2. All specimens were fixed in the 10% formaldehyde solution. 5 General observation On the 1st, 2nd, 3rd, 4th weeks after the surgery, the reaction of incisions was observed and recorded before specimen collection. 6 HE histological observation Specimens were fixed in formaldehyde fluid, embedded by paraffin and sectionst, and then stained with HE. Corium layer, epidermal layer, inflammatory cells, fibroblasts, and collagen on both sides of the incision were observed under light microscopy. 7 Picrosirius-red observation Specimens were fixed in formaldehyde fluid, embedded by paraffin and sectionst, and then stained with Picrosirius-red. The formation of the collagen fiber on both sides of the incision were observed under polarization microscopy. Image Pro-Plus 6.0 was used to analysis the total amount of collagen and the proportion of type I/III. 8 Statistical analysis Statistical analysis was performed with SPSS 21.0 software. All data were presented as Mean±SD. SNK-q test was applied to compare the data of the three groups. P<0.05 was considered as the level of statistical significance.Results: 1 General observationAfter one week of surgery,there was healing wound with clean surface and some stitches fell off and slight sutures reaction in group A and group B. There was the wound covered with seepage, red skin around and some stitches fell off. After two weeks of surgery, there was the healing well wound with flat and similar to the surrounding normal tissuse in group A. There was the healing wound with little sunken in Group B. There was the wound with the obvious healing line and clear edge with the tissuse surrounding in group C. After three and four weeks of surgery, the healing line was not obverious and without growth of fair in group A and group B while the healing line was obvious without growth of fair in group C. 2 Histological observations 2.1 HE staining observation At one week after operation: there was healing skin incision with not obvious keratinization of epidermis, not obvious acanthocyte layer, intact basal layer with regular cell arrangement, regular and loose fiberblast with large cell body and round nucleole, and a few of infiltrated inflammatory cells in group A and group B. There was not healing dermis with obvious keratinization, obvious acanthocyte layer, fiberblast with large cell body, and infiltrated inflammatory cells in group C. At two weeks after operation: there was healing skin incision in three groups. There was flat epidermal layer like surrouding normal tisssue, a lot of fiberblast with larger nucleole, slight fiber bundle paralleled to incision line, and narrow healing line in group A. There was a little sunken epidermal with not obvious keratinization, new slight fiber bundle with loose arrangement like normal tissue, and denser fiberblasts in group B. There was obvious keratinization of epidermis, sunken epidermal, new light in color collagen with vertical arrangement to healing line, and infiltrated inflammatory cells in group C. At three weeks and four weeks after operation: there was a few fiberblasts and loose collagen with alight bundle arrangement like normaltissue in group A and group B. There was more keratinization and denser collagen arraged in bundles. 2.2. Picrosirius-red observation At one week after operation, there was more new green slight type III collagen on two side of incision in group A and group B while there was more red and yellow type I collagen and more black area on two side of incision in group C. At two weeks after operation, there was a lot of green type III collagen on two side of incision in group A. There was less green type III collagen on two side of incision in group B while there was red and yellow type I collagen on two side of incision in group C. At three and four weeks after operation, there was a lot of green type III collagen on two side of incision in group A and group B while there was a lot of red and yellow type I collagen with irregular arrangement on two side of incision in group C. 3 Quantitative analysis of collagen fiber 3.1 The total amount of collagen fiber At one week after operation, the total amount of collagen fiber in group B was the most(62.97±1.21), the amount in group A(62.7±1.29) the second, and the amount in group C(56.34±1.31) the lest. There was significant difference between group A and group C, group B and group C, respectively(P<0.05). But there was not significant difference between group A and group B(P>0.05). At two weeks after operation, the total amount of collagen fiber in group A was the most(70.90±1.26), the amount in group B(69.80±1.29) the second, and the amount in group C(67.26±0.72) the lest. There was significant difference among group A, group B, and group(P<0.05). At three and four weeks after operation, the total amount of collagen fiber in group C was the most, the amount in group B the second, and the amount in group A the lest. There was significant difference among group A, group B, and group(P<0.05).3.2 The ratio of collagen I/III At one week after operation, the ratio of collagen I/III in group C was the most(0.399±0.081), the amount in group B(0.245±0.085) the second, and the amount in group A(0.245±0.084) the lest. There was significant difference between group A and group C, group B and group C, respectively(P<0.05). But there was not significant difference between group A and group B(P>0.05). At two, three, and four weeks after operation, the ratio of collagen I/III in group C was the most, the amount in group A the second, and the amount in group B the lest. There was significant difference among group A, group B, and group(P<0.05).Conclusions: 1 CGF liquid can prmote the healing of incision and reduce inflammation reaction. 2 CGF liquid can increase the total amount of collagen fiber in the early stage of wound healing. 3 CGF liquid can increase the amount of collagen type III in local incision, reduce the ratio of collagen I/III and formation of scar. |
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