| BackgroundAmong war wounds and traumas commonly seen in daily life, tissue defect can be commonly seen in penetrating wounds, amputation wounds, avulsion injury of skin and sever burn. The key point for repairing tissue defect is early successful tissue coverage, which is also an important research subject in battle surgery and in plastic surgery. Fascio-cutaneous flap is one of the most commonly used flaps for repairing soft tissue defect. However, with shallow vein system, it has no blood supply from a named artery which results in the ratio of length to width of flaps in body limited only by 2.5-3 to 1, leading to its restricted clinical application and development. Thus, it becomes a research hotspot how to increase the survival rate of fascio-cutaneous flap with a big ratio of length to width. Currently, definite effects have been proved of VEGF and FGF-2 in enhancing survival of ischemic flap, namely, advancing the pedicle breakage of pedicle skin flap, strengthening the delayed effect of flap, shortening the cycle of flap expansion and resisting retraction after operation. The major effect of VEGF is inducing the formation of capillary vessel, while FGF-2 enhancing the formation of arterioles. It has been reported that VEGF and FGF-2 has synergetic effect, with VEGF being the basis of FGF-2's activation, and FGF-2 enhancing the VEGF's expression, so their combinative utility could bring about the maximized effects in promoting formation of vessels. Under the same in vitro conditions, cellular proliferative amount with the use of VEGF and FGF-2 in combination is 2-8 times more than that with the respective use of either one. These days, the study on using VEGF in combination with FGF-2 is just in the preliminary stage, leaving much space for the development regarding this issue which has great value in clinical application. UMTDT is a atraumatic method and able to avoid or alleviate adverse reaction brought about by systematic administration and also avoid such local side effects as sore pain caused by localized injection. Because of poor blood supply in flap twig, oral and vessel drug administration probably fail to make drugs effectively reach the sites. However, we can take advantage of UMTDT to help drugs of relatively small amount directly reach the ischemic site and attain as well as maintain blood drug level, which is able to accelerate the vessel formation in the ischemic site and the close up of flap especially the ischemic flap. ObjectiveTo validate the effects of VEGF and FGF-2 either in combinative utility or single utility via UMTDT on the survival rate of fascio-cutaneous flap with big ratio of length to width and its microcirculation in pigs. This research will lay a firm experimental foundation for large scale clinical application of fascio-cutaneous flap with big ratio of length to width.Materials and Methods:On the back of ten pigs, we performed 3cm x15cm fascio-cutaneous flaps which were vertical to body's longitudinal axis and on each side divided into 5 treatment groups as follows. A: control group with blank ultrasound; B: VEGF by low-frequency UMTDT; C: FGF-2 by low-frequency UMTDT; D: VEGF and FGF-2 by localized injection in multiple points in ischemia sites three times one day; E: VEGF and FGF-2 by low-frequency UMTDT. With power of 20kHz/3W/cm2, low-frequency ultrasound was performed 15 minutes once, three times per day. All the above treatments lasted five days. After surgery, observations were carried out every day in regard to the color, texture, temperature and swelling etc. Ten days later, judgment of flap survival rate was made and pictures of flaps were transmitted into computer to measure flap survival sizes with the help of computer image analysis system. Then, tissue samples were cut down, fixed with formalin, embedded in paraffin, stained with hematoxylin and eosin and finally made into slices which were used for observing histologic changes via light microscope. Via computer automatic image analysis system we get the data of vessels'intensity in per square centimeter tissue. By immunohistochemistry fluorescence staining with regarding antibody, qualitative and quantitative analysis of VEGF and FGF-2 was made via positive index (Positive index = positive size x OD).Results:1. General observation: Among five groups, as to the necrotic size, A group is the top one, followed by group B and C, and the least belongs to group E. The necrotic sizes near remote end flap for group D and E are less than those of group B and C.2. The survival size and survival rate of flaps: The average survival sizes for five groups are as follows: group A-(25.90±2.60)cm2 ; group B-(34.99±2.91)cm2; group C-(34.58±4.13)cm2; group D-(37.39±4.01cm)2; group E-(40.02±3.33)cm2. The average survival rates for five groups are as follows: group A-(57.55±5.78)%; group B-(77.76±6.47)%; group C-(76.85±9.18)%; group D-(83.08±8.91)%; group E-(88.93±7.41)%. The statistical value of F for survival size and survival rate of flaps is 47.45(P<0.001), which means significant differences among these groups. Further statistical analysis shows that compared with control group (A), all four experimental groups (B,C,D,E) have higher survival size and rate (P<0.001); There is no significant difference between groups B and C with either medication via UMTDT(P=0.708); The both data for injection group combined with UMTDT group with both VEGF and FGF-2 (D + E) are significantly higher than UMTDT groups with either medication(B and C) (P<0.05); Compared with any other group, it is the same case for combinative medication groups (D and E) (P<0.001), with UMTDT(E) more efficient than injection (D) (P<0.05).3. Capillary intensity: For the proximal 1/3 end of the flaps, there exists no significant difference between groups (P=0.071); for the middle 1/3 of the flaps, all four experimental groups higher than control group (A)(P<0.001); There is no difference between single medication groups via UMTDT (B and C)(P=0.990); Injection group and UMTDT group with both VEGF and FGF-2 (D and E) significantly higher than UMTDT groups with either medication(B and C) (P<0.05); Compared with any other group, it is the same case for combinative medication groups (D + E) (P<0.001), with no difference between group D and E(P=0.059); For the far 1/3 end, flaps of control group(A) nearly are all necrotic, resulting in data absence; There is no significant difference between group B and C (P=0.323); The capillary intensity for combinative medication groups (D + E) are greatly higher than any other group(P<0.001); Combinative injection group (D) higher than VEGF group via UMTDT (B)(P<0.05), with no significant difference compared with FGF-2 group via UMTDT (P=0.064); Combinative medication via UMTDT (E) obviously higher than any other group (P<0.001).4. Immunohistochemisty and fluorescent stainingPositive index for VEGF: Compared with control group (A), all four experimental groups (B,C,D,E) have higher positive index for VEGF (P<0.001); There is no significant difference between groups B and C with either medication via UMTDT(P=0.060); The data for injection group and UMTDT group with both VEGF and FGF-2 (D and E) are significantly higher than UMTDT groups with either medication(B and C) (P<0.001), with UMTDT(E) more efficient than injection (D) (P<0.001).Positive index for FGF-2: Compared with control group (A), all four experimental groups (B,C,D,E) have higher positive index for VEGF (P<0.001); There is no significant difference between groups B and C with either medication via UMTDT(P=0.263); The data for injection group and UMTDT group with both VEGF and FGF-2 (D and E) are significantly higher than UMTDT groups with either medication(B and C) (P<0.001), with UMTDT(E) more efficient than injection (D) (P<0.001).Conclusion:All the above research works and statistical analysis are able to reach the following conclusion:1. Administration of VEGF or FGF-2 via UMTDT in ischemic site of fascio-cutaneous flap can significantly increase its capillary intensity; Administration of both medication can further intensify such effects.2. Local administration of VEGF or FGF-2 could significantly enhance both expression, showing their synergic effects to each other. Combinative medication via injection or low frequency ultrasound will further increase positive expression level, with the latter one bringing about longer medication period, lasting effective blood drug level and higher positive expression.3. The medication of VEGF or FGF-2 via GMTDT can greatly increase the survival rate of fascio-cutaneous flaps with big ratio of length to width in pigs. Furthermore, the combinative administration of both VEGF and FGF-2 via injection or low frequency ultrasound is able to further enhance the survival of flaps,with GMTDT group better than injection group.
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