Reversing the detrimental effect of adriamycin on skin grafts in rats

We evaluated in a rat model the effects of a homologous fibrin glue in reversing the effects of Adriamycin on adherence and take of skin grafts. A total of 40 male Fisher rats were used in the study. During the first phase of the experiment, the animals were assigned to either group I (N = 10) receiving normal saline or group II (N = 10) receiving 6 mg/kg Adriamycin by tail vein injection 24 hours before surgery. Skin grafts with and without fibrin glue were placed over wounds in the backs of the animals and adherence was measured at 24 and 48 hours. In the second phase (N = 20), the experiment was repeated, this time evaluating the total area of skin graft take at 7 days. Fibrin glue was found to increase adherence and take of skin grafts in all Adriamycin-treated animals.     

The effect of fibrin glue on skin grafts in infected sites.

Fibrin bonding of skin grafts to wounds is an essential part of the graft-adherence process. Bacteria, in concentrations greater than 10(5)/gm of tissue, are associated with graft failure. Sixty-five rats were randomly divided into three groups, dorsal split-thickness skin grafts were harvested, and the sites were inoculated with Staphylococcus aureus. After incubation, each wound was quantitatively biopsied and treated with saline, fibrin glue with aprotinin, or fibrin glue alone. We found that the addition of commercially available fibrin glue with or without the antifibrinolytic agent aprotinin is capable of restoring graft adherence to normal levels in graft sites infected with greater than 10(5) bacteria/gm of tissue. Fibrin glue may have potential for increasing skin-graft take in the clinical situation where the graft bed is infected.     

The lack of effect of pentoxifylline on random skin flap survival

The effects of pentoxifylline on skin flap survival were studied in rabbits. A total of 40 rabbits had caudally based single-pedicle flaps measuring 4 x 14 cm raised on the mid dorsum of each animal. Twenty of these rabbits were given intraperitoneal injections of pentoxifylline in doses of 24 mg/kg per day beginning 48 hours prior to flap construction and continued daily for 7 days postoperatively. The remaining 20 control rabbits received intraperitoneal injections of saline in equal volumes as the experimental groups. At the end of 7 days, viable flap length was visually inspected and measured in all 40 rabbits. There was no significant difference in skin flap viability in rabbits treated with pentoxifylline compared to the control group.     

Skin graft survival after external beam irradiation.

Difficulties with skin graft ulceration after radiation therapy for cancer have led many to question the suitability of this method of soft-tissue coverage and its cost-effectiveness. The objective of this study was, therefore, to assess skin-graft integrity subjected to postoperative external beam irradiation in a rat model. The model consisted of a rectangular full-thickness skin graft raised and reapplied to its original bed on the dorsum of each rat. Five groups of adult male Sprague-Dawley rats (n = 8 per group) were established. Group A was the control group and was not given postoperative irradiation. Groups B, C, D, and E received postoperative unfractionated cobalt60 irradiation 4 weeks after grafting for a total dose of 15, 20, 25, or 30 Gy, respectively. Weekly skin-graft evaluation was performed for the 4 weeks after irradiation (8 weeks after surgery) by measuring areas of graft loss using computerized planimetry. After the animals were killed, histologic samples were obtained from normal unirradiated skin and from both intact and ulcerated skin-graft sites. Graft loss after irradiation of < or = 20 Gy was similar to that of the unirradiated controls. Occurring as early as 1 week after treatment, a two-fold increase in graft ulceration was observed with doses of > or = 25 Gy (p = 0.0007). Only partial healing of ulcerations was noted by the fourth week after treatment. Histologic changes associated with the irradiation of skin grafts using doses of 25 Gy or higher included hyaline degeneration, fibrinoid necrosis, telangiectasia, and edema. Granulation tissue predominated as a mechanism of healing in areas of graft ulceration. The intensity of inflammatory cell infiltrate did not correlate with radiation dose. The authors concluded that postoperative, unfractionated irradiation can induce skin-graft loss at doses of 25 Gy or higher. Fractionated irradiation or longer intervals between grafting and irradiation may increase skin-graft tolerance; however, further studies are warranted.     

Use of free thin anterolateral thigh flaps combined with cervicoplasty for reconstruction of postburn anterior cervical contractures

Free thin anterolateral thigh flaps combined with cervicoplasty were used in a series of seven patients undergoing reconstruction for previous burn injury from September of 2000 to May of 2001 at Chang Gung Memorial Hospital. This method uses a suprafascial dissection technique to provide a thin flap to improve cervical contour. Neck contractures had resulted from flame burns in six patients and from a chemical burn in one patient. The mean age was 32.7 years (range, 22 to 45 years). The size of excised scar ranged from 10 x 2 cm to 26 x 5 cm (mean, 19.7 x 3.3 cm). The size of flaps ranged from 11 x 5 cm to 26 x 8 cm (mean, 21.3 x 6.5 cm). Average operative time was 6 hours. Average hospital stay was 10 days. All flaps survived, with one flap sustaining partial marginal loss. The donor site was closed primarily in five cases and by using a split-thickness skin graft in two cases. At a mean follow-up time of 5 months, the functional improvement was measured as follows: a mean increase in extension of 30 degrees (preoperatively, 95 degrees; postoperatively, 125 degrees), a mean increase in rotation of 18 degrees (preoperatively, 59 degrees; postoperatively, 77 degrees), and a mean increase in lateral flexion of 12.5 degrees (preoperatively, 26.5 degrees; postoperatively, 39 degrees). The average cervicomandibular angle was improved by 25 degrees (preoperatively, 145 degrees; postoperatively, 120 degrees). This series demonstrates that the use of free thin anterolateral thigh flaps combined with cervicoplasty provides a one-stage reconstruction with a thin, pliable flap that achieves good cervical contour with low donor-site morbidity.     

Possibility of predicting rat wound epithelization by changes in matrix metalloproteinases activities in wound exudate

The engraftment of a free skin graft introduced into an unhealing wound as a source of epithelization in combination with a transplantation of a dermal equivalent was studied in rats. The course of wound healing was estimated by changes in the activity levels of metalloproteinases (MMPs) in wound exudates. It was shown that the results of skin-graft transplantation could be predicted by monitoring changes in wound exudates MMP-2 and MMP-9 activities. It was found that engraftment of the skin graft occurred at intermediate activity values of MMP-2 and MMP-9 in the wound exudates, whereas their low and high activities correspond to lysis of the transplanted skin graft.     

Drug treatment and flap survival

Some investigators found that isoxsuprine, propranolol, or heparin would increase skin-flap survival in loose-skinned animals. We evaluated the effects of these three drugs in the pig, an animal with skin circulation similar to that of humans. Four hundred ventrally based skin flaps that have a proximal axial portion and a distal random portion were made on the flanks of 40 pigs. There were eight study groups: control, isoxsuprine preoperatively and postoperatively, propranolol preoperatively and postoperatively, isoxsuprine postoperatively only, propranolol postoperatively only, heparin, single-stage surgical delay, and two-stage surgical delay. Flap survival was improved by the two-stage surgical delay when compared with the control flaps, flaps from pigs receiving a drug, or flaps from pigs having a single-stage surgical delay (p less than 0.001). When compared with the control flaps, neither isoxsuprine, propranolol, heparin, nor single-stage surgical delay significantly increased flap survival.     

Fluorometric analysis of an attempt to reclaim ischemic flaps in rats with Fluosol

An experiment was designed to answer two questions as they apply to random skin-flap survival: Is there a therapy that can improve random skin-flap survival when given postoperatively? And if so, when does one start such a therapy? Fluosol-DA 20% (Fluosol) has increased random skin-flap survival when given preoperatively in our laboratory. An experiment was devised to see if it could rescue failing flaps. One-hundred Sprague-Dawley rats were divided into a control (N = 25) and five experimental groups (N = 15). All had 10 X 13 cm reverse McFarlane random flaps raised and reinset. The experimental groups underwent hemodilution with either Ringer's lactate or Fluosol at 4, 8, and 12 hours after flap elevation. All were kept in 50% oxygen for 72 hours postoperatively. The flaps and their corresponding necrotic areas were measured on day 7. As to when to institute a therapy, we simultaneously evaluated the use of a microfluorometer as a monitor of flap survival. Analysis of flap survival showed little difference between control and experimental Ringer's lactate or Fluosol groups. Analysis of the microfluorometric data led to the following points. First, as a monitor of flap viability, it is limited by a lack of specificity and sensitivity. Second, comparison of the data from portions of the flap destined to live with those destined to die suggests that it may not be failure of circulatory inflow that leads to flap death.     

Effect of liposuction on skin perfusion

Clinical reports of full-thickness skin necrosis have raised concern about the thermal and dermal ischemic effects of ultrasound-assisted liposuction. The purpose of this study was to evaluate skin perfusion in patients treated with ultrasound-assisted liposuction or suction-assisted liposuction. Patients (n = 75) were studied prospectively in the perioperative period surrounding their suction-assisted liposuction (31 patients) or ultrasound-assisted liposuction (64 patients). The laser Doppler flowmeter was used to monitor skin perfusion in the treated regions preoperatively, intraoperatively, and postoperatively at a series of time intervals. The effects of the anesthetic, wetting solution, and type of liposuction (suction-assisted liposuction or ultrasound-assisted liposuction) on skin perfusion were measured. Anesthetic induction significantly increased measured skin perfusion. Wetting solution infusion significantly decreased skin perfusion (-57.4 percent +/- 2.0) by 15 minutes postinfusion. Skin perfusion in the ultrasound-assisted liposuction group was significantly greater than that of the suction-assisted liposuction patients at 1 hour, 1 day, and 1 week postoperatively; however, by 2 to 5 weeks, no difference in skin perfusion was noted and skin perfusion had returned to preoperative levels in both groups. Although skin perfusion in the suction-assisted liposuction group was significantly lower than in the ultrasound-assisted liposuction group in the early postoperative period, no differences in skin perfusion between the groups were noted beyond 1 week postoperatively, suggesting that neither technique impairs perfusion.     

The effect of epinephrine in local anesthesia on the survival of full- and split-thickness skin grafts: an experimental study

The effect of local anesthesia containing epinephrine on the survival of split- and full-thickness skin grafts remains unclear. In this blinded study, Xylocaine with or without epinephrine was injected subdermally prior to harvesting of split-thickness and full-thickness skin grafts on the dorsum of rabbits. After procurement, the grafts were placed back into their original donor sites. Statistical analysis of graft survival 7 days postoperatively revealed a significant decrease in survival for the full-thickness skin grafts treated with Xylocaine with epinephrine as compared with similar grafts without epinephrine (p less than 0.0005). No significant difference was noted for split-thickness skin-graft survival in grafts treated with Xylocaine with and without epinephrine (p greater than 0.1).     

Evaluation of the mechanism of vascular endothelial growth factor improvement of ischemic flap survival in rats

This study evaluated the effects of exogenous vascular endothelial growth factor (VEGF) on the regulation of cytokines in a rat dorsal ischemic skin flap model. Exogenous VEGF (1 microg/ml) was injected subdermally into the flaps of 12 rats before the flaps were sutured back in place. Another 12 rats with flaps received saline injections, as a control group. Biopsy specimens were obtained from the flaps treated with VEGF or saline solution, at positions 2.5, 5.5, and 8.5 cm from the distal edge of the flaps, at 12 hours (n = 6 for each group) and 24 hours (n = 6 for each group) after suturing of the flaps. Expression of cytokine, growth factor, and inducible nitric oxide synthase was measured. The results demonstrated that expression of tumor necrosis factor-alpha and nitric oxide synthase in the distal part of the VEGF-treated flaps was significantly decreased, compared with the control values, at 12 and 24 hours postoperatively. It was concluded that administration of exogenous VEGF could protect flaps from ischemia-reperfusion injury through the regulation of proinflammatory cytokines and the inhibition of cytotoxic nitric oxide production.     

Enhanced survival of full-thickness skin grafts following the application of DC electrical fields

The present study was undertaken to determine if uses of exogenous electrical fields could improve the posttraumatic quality of dermis and epidermis in isolated grafts in the rat. In blinded procedures, a full-thickness area of skin was removed and reattached to the original site. A galvanic device delivering 4.5 microA of direct current was applied using three electrode orientations: (1) anode above graft (AT; N = 8), (2) cathode above graft (CT; N = 7), and (3) no current (NC; N = 7). Current delivery was discontinued 4 days postoperatively. Quantitative assessment at 7 days postoperatively indicated the presence of necrotic skin over 80 to 90 percent of the graft surface area in NC and CT animals. In contrast, only 50 percent of the graft area was necrotic in AT rats. Histologic examinations indicated a significantly thickened dermis in the AT (versus NC and CT) rats, accompanied by patches of multilayered intact epidermis, which was virtually absent from the other experimental groups. Results are consistent with the hypothesis that direct-current stimulation can affect skin graft survival and repair.     

Increase in skin-flap survival by the vasoactive drug buflomedil

The effect of buflomedil to protect skin tissue from ischemia and necrosis was studied in random cutaneous flaps. Measurements were performed by intravital microscopy on the microcirculatory level of capillary perfusion in a flap model in the hairless mouse. In 30 hairless mice, single-pedicle flaps measuring 6 x 16 mm were raised perpendicular to the spine of the animal. This flap develops a reliable amount of necrosis at its distal edge over a period of 7 days. A group of 10 mice received intravenous injections of buflomedil in doses of 3 mg/kg per day diluted in 0.1 ml normal saline beginning 4 hours before flap elevation and for 6 consecutive days postoperatively. In addition, 10 further animals received the same treatment except that it was started 5 minutes after flap elevation. In 10 mice serving as controls, normal saline in equal volumes as in the experimental groups was applied. By means of intravital microscopy, functional vessel density (FVD) was determined in 2.5-mm increments from the flap's base to its distal edge at 1, 6, and 24 hours after elevation. Skin-flap survival was quantified by measuring the necrotic area on day 7 by means of digital planimetry. Functional vessel density was preserved in the distal flap of animals pretreated with buflomedil, revealing a higher functional vessel density at 10.0 mm (p less than 0.01), 12.5 mm (p less than 0.05), and 15.0 mm (p less than 0.001) from the flap's base as compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of glucocorticoid treatment on skin capillary blood flow and viability in cutaneous and myocutaneous flaps in the pig

The effect of methylprednisolone treatment on skin-flap viability and capillary blood flow was studied in a series of four experiments. Intramuscular methylprednisolone injections (30 mg/kg per day), given in single or divided doses preoperatively or postoperatively, had no effect in augmenting skin viability in arterialized cutaneous, myocutaneous, or random skin flaps compared with the control. Capillary blood flow was studied in arterial buttock flaps and latissimus dorsi myocutaneous flaps raised on animals treated preoperatively with methylprednisolone or saline (control), and no significant difference in capillary blood flow was noted between the treatment and control flaps. It was concluded that methylprednisolone has no significant therapeutic effect either in increasing flap viability or in increasing capillary blood flow in skin flaps in pigs.     

Preparation and evaluation of a nonproprietary bilayer skin substitute.

Cross-linked, allogeneic, telopeptide-depleted dermal grafts were lyophilized and laminated with silicone rubber elastomer. Resultant bilayers were studied for incorporation into the wound site and capacity to inhibit cutaneous wound contraction in experimental animals. Bilateral full-thickness skin wounds were made in 20 male New Zealand white rabbits. One side was grafted with the processed graft, while the contralateral side remained ungrafted as a control wound. Over 63 days, wound sites were analyzed at intervals on the basis of the extent and rate of wound contraction and by histologic examination. Cutaneous wounds successfully incorporated graft matrix and were significantly inhibited in their rate and extent of wound contraction. Notably, by day 63, grafted wounds retained 71 percent of their original area, whereas ungrafted control wounds retained only 16 percent of their original area. There were no graft rejections, and the bilayer graft's dermal analogue appeared to function as a biodegradable template that physically conformed neodermis to a preestablished pattern while counteracting contractile forces. This investigation suggests that, in experimental animals, the success of bilayer dermal grafts is less dependent on highly specialized and complex preparative techniques than typically has been presumed and that relatively simple, previously published, nonproprietary techniques, when adapted to a bilayer format, yield acceptable results as defined in terms of biocompatibility, capacity for graft incorporation, and inhibition of wound contraction.     

Therapeutic value of intravenous heparin in microvascular surgery: an experimental vascular thrombosis study

In an attempt to decrease a 10 to 15 percent vascular thrombosis rate leading to graft occlusion, low-dose human-grade heparin was studied to determine if carefully monitored intravenous therapy would increase 7-day patency in a known potent thrombosis model. In New Zealand white rabbits, the type of infusate administered intravenously, either saline (30 animals) or heparin (35 animals), was selected at random after completing a 2-mm arterial inversion graft in the femoral artery. A 72-hour infusion was used in all animals; the control group received sterile saline and the experimental group received a heparin infusion at 45 microliters per hour after a 500-unit bolus. All grafts in both groups were patent at the time of groin closure. Patency in the heparin-perfused group was 67 percent (24 of 35) as compared to 19 percent (6 of 30) in the control group (p less than 0.05) 1 week postoperatively. Scanning electron microscopy showed significantly less dense fibrin deposition and a decrease in the number of aggregated platelets in the heparin-perfused grafts. Partial tissue thromboplastin time values in the experimental group ranged between 55 and 75 seconds (control 20 to 25 seconds). We have shown that heparin, an inexpensive and readily available agent, maintains 1-week microarterial patency and results in few complications in a reliable, reproducible, and versatile thrombosis model. The clinical ramifications of using an antiplatelet agent that diminishes fibrin deposition in microsurgery are apparent.     

The influence of systemic growth hormone administration on the healing time of skin graft donor sites in a pig model

A more rapid healing of skin graft donor sites has often been observed during ultimoratio therapies with growth hormone in adults who have suffered extremely severe burns. The purpose of this animal experimental study was to examine the influence of systemic growth hormone administration on the healing time of skin graft donor sites under standardized conditions in pigs. The animals were 14 (7 experimental and 7 control) male, sexually mature, German domestic pigs, in which 30 skin graft donor sites 8 cm x 4 cm and 0.6 mm deep were created. Fifteen each of the skin graft donor sites were bandaged with the same material [hydrocolloid bandage (Varihaesive E) and PVP-iodine gauze (Braunovidon Gaze)]. The test period was 15 days for each pig, whereby recombinant growth hormone (0.5 IU/kg body weight per day) was applied subcutaneously in the experimental group. The bandages were changed under brief narcosis every 2 days, during which one skin-punch biopsy was taken per skin graft donor site, and blood samples were drawn for determination of the serum IGF-1 values. Photographic documentation was also recorded. The biopsies were examined histologically (hematoxylin and eosin stain) and immunohistochemically (collagen IV and VII, and laminin), whereby histologically the start of keratinization was assessed as a healing criterion. The serum IGF-1 values in the growth hormone group were statistically significantly higher than in the control group. Immunohistochemically, a complete basal membrane was observed in both the experimental and the control group after the 7th or 8th day. A clearly elevated serum IGF-1 level correlated in the growth hormone group with the skin graft donor sites healing. It could thus be demonstrated both clinically and histologically that systemic application of growth hormone results in a statistically significantly more rapid healing of the skin graft donor sites by 2 days earlier than in the control group.     

When does a random flap die?

A random flap can be constructed, its circulation determined, and the ischemic portion identified. Left untreated for a period, the critical ischemia time, the ischemic portion will die and is clinically recognized several days later. What is not known is when this tissue, destined to die, actually dies. To ascertain this time, we compared the percent necrosis of a distal 3 x 3 cm segment of a 10 x 3 cm reverse McFarlane random flap with a known distribution of necrosis to the percent necrosis of the distal 3 x 3 cm of full-thickness skin grafts taken from a similar reverse McFarlane flap at 0, 4, 8, 12, and 16 hours after pedicle construction. Implicit in this experiment is the assumption that necrosis of the full-thickness skin grafts in excess of that of control animals represented skin no longer viable. Sometime between 8 and 12 hours, the percent necrosis of the full-thickness skin grafts surpassed that of the control, and it was concluded that this graft was dead prior to grafting. Thus it is suggested that critical ischemia time and death of the flap tissue are nearly identical, and the latter occurs at between 8 and 12 hours.     

The critical relationship between free radicals and degrees of ischemia: evidence for tissue intolerance of marginal perfusion

Skin-flap ischemia has been associated with the presence of free radicals. In this study, two enzyme systems involved in free-radical metabolism were used to compare a distal skin flap to a skin graft. Forty-two rats were divided into several test groups. A 10 X 3 cm dorsal rat flap was used, and tissue biopsies for xanthine oxidase and malonyldialdehyde (MDA) were obtained 2.5, 5.5, and 8.5 cm from the base of the flap at the hours given. In group I (control), the flap was outlined but not elevated, and biopsies were obtained. In group II, the flap was elevated, and biopsies were obtained at 6 hours. In group III, the flap was elevated, the distal 4 X 3 cm was amputated and replaced as a full-thickness skin graft, and biopsies were obtained at 6 hours. In group IV, the flap was elevated, and biopsies were obtained at 12 hours. In group V, the flap was treated as in group III, and biopsies were obtained at 12 hours. In group VI, the flap was elevated, and biopsies were obtained at 24 hours. In group VII, the flap was treated as in group III, and biopsies were obtained at 24 hours. Results: Xanthine oxidase was significantly higher in all distal biopsies compared to proximal biopsies. Xanthine oxidase also increased with time. Malonyldialdehyde increased over time as well as with distance from the flap base. Distal flap biopsies at 24 hours had greatly increased levels of malonyldialdehyde compared to skin grafts (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental and clinical applications of fibrin glue

A 2-year experience with laboratory and clinical applications of fibrin glue is presented. An autologous technique, which eliminates the danger of multidonor preparations, has been developed in our blood bank. While one can obtain different fibrinogen concentrations from the same amount of a patient's blood, in vitro mechanical testing demonstrated that at higher fibrinogen concentrations there is an increase in shear adhesive strength. Evaluation of skin-graft take in 16 Sprague-Dawley rats did not demonstrate significant differences in healing when adhesive use was compared with suture technique. In a clinical study, four different groups of patients (facial burns, hand burns, difficult graft sites, and miscellaneous surgical applications) benefited from autologous or single-donor fibrin glue for a total of 82 cases. There are several distinct advantages to the use of fibrin adhesive: The autologous technique eliminates the risk of transmissible viral diseases (AIDS, hepatitis); it can be used as a sealant in the treatment of seromas, dural leaks, and lymphoceles; and it improves hemostasis and early graft adherence. Face and hands are resurfaced with sheet grafts in a single procedure, obtaining a better aesthetic result with complete graft take and immediate start of physical therapy. Neither sutures nor pressure dressings are required. The minimal postoperative care associated with early return to normal activities seems to increase the satisfaction of patients and nurse personnel.