Abstract
The aim of this study was to compare vacuum and conventional dressings and to follow revascularization with laser-Doppler spectroscopy and histological evaluations. In six minipigs, full thickness skin grafts were harvested on both sides of the back and transplanted to the contralateral site. One site was covered with a classical cotton dressing, the other with a vacuum dressing. For 10 days, oxygen, haemoglobin, flow and velocity levels were measured by laser-Doppler spectroscopy. Data were analyzed with ANOVA. Histological evaluation included haematoxylin and eosin (H&E) as well as CD31 immunohistochemical staining. Oxygen levels were significantly reduced in the vacuum dressing group compared with the classic dressing group during the first 3 days after transplantation. Haemoglobin levels were slightly, but significantly, higher in the vacuum dressing group over the whole observation period. On the second and third day after transplantation single capillaries were detectable in the histological evaluation. Starting from the fourth day, capillary number increased. Similar results for the classical pressure dressing and the vacuum dressing were observed. The present experimental model provides a standardized and reliable test system for evaluating revascularization of full thickness skin grafts in conjunction with growth factors and other enhancers of revascularization.
Full thickness skin grafts are needed every day for reconstructions, for example to cover defects after tumour surgery or to save lives after burn injuries. Few controlled studies have been published evaluating different dressings during the first days, even though they play a key role in the success of surgery . Existing knowledge about the healing process of full thickness skin grafts is based on studies conducted several years ago with the resources available at that time: microscopy , microangiography and thermography . More recent examinations with new technologies are lacking.
During the first days after transplantation, full thickness skin grafts are supported by diffusion from the wound bed (imbibition) . Attachment to the bed is by fibrin . After 2–3 days small anastomoses between bed and transplant begin to form (inosculation) . Full circulation is complete within 4–7 days . If revascularization fails after about 5 days, the transplant fails. This can be due to missing contact through seroma formation between transplant and bed . To reduce this danger, compression dressings are recommended . Sutures can be used to fix a compression dressing , but this can cause varying pressure or ulcers. Vacuum dressings might unify the pressure distribution. Tie-over dressings offer no visual wound control or possibilities for correction. The course of healing of full thickness skin grafts is mainly judged clinically without quantifying it in an objective manner. Complications may be detected too late, complicating treatment and impairing prognosis.
Laser-Doppler spectroscopy combines well established methods (spectroscopy and laser-Doppler ). One advantage is the analysis of all parameters at one time and in one place, thus saving time. This is important as the values can vary even in close sites . Postoperative transplant controls for evaluation of the blood supply in deeper levels were performed successfully with this system .
The hypothesis of this study was that vacuum dressing results in earlier revascularization and better healing of skin grafts compared with conventional cotton dressings.
Material and methods
Six minipigs were used in this study. The protocol was in accordance with the German animal welfare act (Animal Experiment Permit V-742-72241.121-14 (39-5/04)). All animals received food and water ad libitum .
Surgery was carried out under general anaesthesia. All animals received single-shot penicillin (5 M) i.v. before surgery. Their backs were shaved, then disinfected with povidone iodine. After harvesting full thickness skin grafts with scalpel and forceps, residual fat tissue was removed carefully with scissors down to the dermis. Transplants were sutured on the contralateral site after being meshed with scalpels. On the left side, a classic tie-over bolster cotton dressing with iodine was fixed with long sutures, on the right side, a vacuum dressing (VAC with white sponge, KCI Medizinprodukte, GmbH, Wiesbaden, Germany) was applied. A vacuum-pump with a battery was sutured in a bag on the back of the animals. To avoid contamination, the whole back was covered with a cotton fleece and the animals were kept in single boxes. As recommended by the manufacturer, the negative pressure was set to a sub-atmospheric pressure of 125 mm Hg . The next morning, all animals were moving completely normally. Both dressings were removed after 5 days. For the first 7 days, cylindrical biopsies of the full thickness skin graft and the wound bed from two animals were taken. After 10 days, the animals were killed. At the end of the observation period, transplants on both sides were excised together with the surrounding soft tissue.
The transplants were evaluated before surgery, directly after transplantation and then every 24 h with the laser-Doppler spectroscope O2C (Lea Medizintechnik, GmbH, Giessen, Germany) for 10 days. Clinical parameters of wound healing such as swelling and haematoma were documented. The bolster dressings were moved to the side to place the detector directly on the transplants. In the vacuum dressing group, measurements were made through the dressing. The O2C allows the measurement of perfusion and oxygenation at the same time .
The blood flow velocity was the mean velocity of the moving red blood cells, relative blood flow was the mean velocity of the red blood cells related to the number of red blood cells in the measured volume. The O2C combines Doppler flowmetry and tissue spectrophotometry: with a fiberoptic system, laser with a wavelength of 820 nm was directed onto the tissue. The light was reflected from the moving red blood cells causing frequency changes (linear correlation). Analysis was carried out through the same probe. Blood flow was determined by the intensity of reflection. Selective depths could be measured by varying the wavelength and the detector distance. Haemoglobin levels and oxygenation were determined by tissue spectrophotometry. The emitted light was absorbed and scattered causing spectral changes. The spectra were compared with oxygenated and non-oxygenated haemoglobin. As vessels with a diameter larger than 100 μm absorb nearly all the light, capillaries and very small vessels were evaluated in particular. Relative haemoglobin levels were calculated by addition of haemoglobin absorption of all wavelengths .
After stabilization of the flow values after some seconds, the laser-Doppler spectroscopy values were recorded. Levels of haemoglobin oxygenation, relative haemoglobin levels, relative blood flow levels and relative blood velocity were measured in relative units ( www.lea.de ).
Histological assessment of the skin graft tissue was based on light microscopy. Four micrometer thick sections of formalin-fixed, paraffin-embedded tissue from the original skin specimen were stained with H&E. Three micrometer thin paraffin sections were de-paraffinized and rehydrated and immunohistochemical stains with CD31 as an endothelian marker were carried out according to routine methods. Specimens of the full skin graft tissue were analyzed by a pathologist.
To achieve a normal distribution, the oxygen values were transformed following this formula: log(SO 2 /(100 − SO 2 )). The data were analyzed with ANOVAs with the factors ‘group’ (classical tie-over bolster dressing vs. vacuum dressing) and ‘day’ after surgery. The least squared means were calculated and presented in figures (oxygen levels were re-transformed for the figures).
Results
At the end of the observation period, large areas of all transplants showed macroscopic perfusion similar to the surrounding tissue and small parts with haematoma, but no differences were obvious between the classic dressing group and the vacuum dressing group. No transplant was lost and all animals behaved normally.
Results
At the end of the observation period, large areas of all transplants showed macroscopic perfusion similar to the surrounding tissue and small parts with haematoma, but no differences were obvious between the classic dressing group and the vacuum dressing group. No transplant was lost and all animals behaved normally.
Laser-Doppler spectroscopy
Oxygen levels were markedly reduced in the vacuum dressing group during the first 3 days after transplantation (see Fig. 1 ). Afterwards the levels were similar for both groups. The effects for ‘group’ and the combination ‘day × group’ revealed statistically significant differences. Haemoglobin levels were more constant and slightly higher in the vacuum dressing group over the whole observation period ( Fig. 2 ). The differences were statistically significant for all effects tested. The flow levels reduced during the first 3 days in both groups ( Fig. 3 ). Afterwards they increased rapidly for both groups. Starting from the sixth day, the levels of the vacuum dressing group had a higher increase. All effects were statistically significant. For velocity levels, the curves were similar for both dressings ( Fig. 4 ): they decreased for the first 3 days and then increased. The deepest levels in the vacuum group were observed on days 2–3, and in the classic dressing group on days 3 and 4. The effects ‘day’ and the ‘combination’ were statistically significant. Test details for the different parameters evaluated are presented in Table 1 and effect details for the statistical tests by parameter are shown in Table 2 .
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