A Quantitative Analysis of Microcirculation During Healing of Split-Thickness Skin Grafts in Standardized Full-Thickness Wounds

In clinical practice, skin grafts are commonly used in the management of skin defects caused by burns, trauma, or microvascular disease. Skin grafting remains the standard of care and is the second most common method of achieving wound closure after primary wound closure.¹ Skin grafts include split-thickness or full-thickness autologous grafts, as well as allogenous grafts such as decellularized human cadaver skin.^2,3 For large wounds, the use of split-thickness skin grafts is recommended. After debridement of the injured recipient area, a dermatome is used to harvest a thin layer comprising the epidermis and superficial papillary dermis from a different anatomic area, typically the anterior or lateral thigh.⁴ After harvesting, the split-thickness skin graft is usually meshed to achieve coverage of larger areas. 

The physiology behind skin graft revascularization is quite complex; the healing of skin grafts depends on the formation of new vessels in the fibrin interface between the skin graft and the treated wound area.⁵ Scientists began studying the mechanisms behind skin graft revascularization in the 1800s. At that time, it was believed that the graft and host vessels would directly reconnect during graft healing, a process called inosculation.^5,6 In the 1960s, researchers found that endothelial cells of the graft vessels degenerated, leaving only the basement membrane; this provided a potential conduit for the ingrowth of new vessels.⁷ It was also found that the vessel pattern in the graft was the same before and after the skin graft was harvested.⁸ Other researchers found clues that indicated a combination of vascular inosculation as well as vascular ingrowth into the skin graft.⁹ However, the exact mechanisms of skin graft revascularization as well as the timing of this process remained unclear for many decades. More recent studies of transgenic mice have demonstrated that the replacement of graft vasculature through endothelial cells as well as endothelial progenitor cells along preexisting pathways is the main mechanism of skin graft revascularization.⁵ In recent years, there has been increasing awareness of understanding the mechanisms of spontaneous and skin-grafted wound healing as a route to artificial skin replacement.¹⁰

Nevertheless, the formation and remodelling of the vessel architecture in skin grafts remains a fascinating process, because it is complex and crucial to graft healing. The goal of the current study was to evaluate microcirculatory changes during the wound healing process after skin grafting in a rat model within a period of 84 days to determine how and for how long microcirculation and perfusion dynamics change after skin grafting.

Animals

The current study used 14 adult male Lewis rats (Charles River Laboratories) with an initial age of 6 to 8 weeks and a mean (standard deviation) body weight of 280 (12) g. The rats were housed individually, and the experiments were carried out according to current guidelines for the care and use of laboratory animals after the authors received the approval of the local animal care committee.

Anesthesia

Surgery and the subsequent measurements of microcirculation were performed under intraperitoneal anesthesia, with a single injection of a combination of fentanyl (0.005 mg/kg bw), medetomidine (0.15 mg/kg bw), and midazolam (2 mg/kg bw).¹¹ After surgery or measurement, the anesthesia was subcutaneously antagonized with naloxone (0.12 mg/kg bw), flumazenil (0.2 mg/kg bw), and atipamezole (0.75 mg/kg bw).¹¹

Study design

One full-thickness wound including skin and subcutaneous layers with an extend of 2 cm × 2 cm was generated on the cranial part of the back on each rat (n = 14), reaching down to the fascia of the back muscle. A split skin graft with a thickness of 0.3 mm was harvested from the caudal part of the back using a skin dermatome, as described by Rahmanian-Schwarz et al.¹² All wounds were treated with non-meshed split-thickness skin graft transplantation only. The skin grafts were fixed with skin staples, and the wounds were covered with fatty gauze and occlusive foil to prevent contamination.

Evaluation of microcirculation

For quantitative assessment of microcirculation, the O2C device (Oxygen to See; LEA Medizintechnik) was used.^13,14 The device transmits continuous wave laser light at 830 nm (30 mW) and white light at 500 nm to 800 nm (20 mW) to the tissue. The movement of erythrocytes causes a Doppler shift, which is detected by the probe. From this, the O2C calculates the blood flow. 

Through spectrophotometry using a white light that is reflected by erythrocytes at certain wavelengths depending on their SO2, the device determines the hemoglobin oxygenation (percentage) and the rHb in the tissue (AU). All parameters are measured at the same time and via one probe; in the current study, measurements were done at depths of 2 mm and 8 mm.

The measurements were performed by the same researcher and under the same environmental conditions 10, 20, 30, 40, 50, 60, 70, 80, and 84 days after surgery. As a reference value, untreated skin was measured 3 cm away from the edge of the wound. 

Statistical analysis

For statistical analysis, the t test was used to compare values. Statistical significance was set at 5% (P ≤ .05). Analysis was performed with SPSS version 20.0 (IBM Corporation).

Complete wound healing of all split-thickness skin grafts, that is, macroscopic complete epithelialization of the created wounds, was observed within the experimental period of 84 days (Figure 1). Changes in microcirculation (blood flow, SO2, rHb) were quantitatively assessed by means of the O2C device in superficial (2 mm) and deep (8 mm) skin layers.

Blood flow

Blood flow is a parameter that represents the blood supply in the capillaries of the examined tissue. Compared with blood flow in healthy skin, blood flow in all wounds was increased 10 days (first measurement after surgery) after split-thickness skin graft transplantation in superficial and deep skin layers. The median blood flow at a depth of 2 mm in healthy skin was 49 AU, compared with 128 AU after skin grafting (Figure 2). At a depth of 8 mm, median values of 144 AU in healthy skin and 235 AU in grafted skin were measured. Blood flow in the superficial skin layer increased compared with healthy skin until the measurement on day 60, at which time a blood flow of 48 AU was detected in the grafted superficial skin layer. After that time point, superficial blood slightly increased again above baseline values. This trend continued until the last measurement, on day 84. Deep blood flow increased until the last measurement, on day 84, when it first reached the baseline value of healthy skin with a median 144 AU. 

Compared with healthy skin, statistically significant changes in blood flow in wounds were observed from day 10 to day 50 in superficial and deep skin layers.

Oxygen saturation

SO2 is measured in the postcapillary venules and thus is a parameter for the amount of oxygen extraction in the examined tissue. Compared with healthy skin, which demonstrated mean SO2 values of 41% at a depth of 2 mm and 48.5% at a depth of 8 mm, at 10 days after surgery SO2 decreased to 17% in superficial layers but increased to 53% in deep skin layers (Figure 3). In superficial layers, the SO2 remained decreased compared with the baseline of healthy skin until final follow-up on day 84. The lowest value (16%) was recorded on day 20; the value increased slightly afterward. In deep skin layers, a further increase in SO2 was detected until day 40 (63%), after which it slowly decreased again; however, SO2 remained increased compared with the baseline of healthy skin until final follow-up on day 84. 

Compared with healthy skin, statistically significant changes were observed in all SO2 measurements, except on day 10 in deep skin layers and day 80 in superficial skin layers.

Relative amount of hemoglobin

The rHb gives information about the amount of blood in the examined tissue. On day 10, at a depth of 2 mm the mean rHb was elevated (69 AU) compared with healthy skin (58.5 AU) (Figure 4). At a depth of 8 mm, the rHb was elevated as well, with a mean value of 90 AU compared with 66 AU in healthy tissue. At a depth of 2 mm, only the value measured on day 10 was above the baseline for healthy skin. From day 20, the rHb undulated slightly, but it always stayed below the baseline value measured in healthy skin. At a depth of 8 mm, the mean rHb on day 20 in the treatment group was 78 AU, which was still higher than the baseline value in healthy tissue; however, from day 30 on, the mean value in the treatment group remained below the baseline value measured in healthy skin.

Compared with healthy skin, statistically significant changes were observed on all days except day 20, 40, and 50 in superficial skin layers (depth of 2 mm) as well as on all days except day 20, 30, 50, and 80 in deep skin layers (depth of 8 mm).

The values for blood flow measured by the O2C device represent the blood supply in the capillary bed of the examined tissue. The measured capillary venous SO2 reflects the oxygen extraction; the rHb is a parameter for the amount of blood in the examined tissue.¹⁵

It appears that blood flow, SO2, and rHb are higher in deeper layers of tissue. Reference values for microcirculation at different tissue depths in rodents have not yet been established. Human studies have demonstrated that there is a higher amount of blood flow in deeper tissue layers due to an increased density of capillaries and metabolic activity.¹⁶ Considering rodent anatomy with the panniculus carnosus as an additional muscle layer in the subcutis, higher blood flow in deeper tissue layers seems plausible as well.¹⁷ In the grafted wounds in the current study, the deep tissue layers 8 mm beneath the probe were recorded in the midst of muscle tissue as the created wounds reached the muscle fascia, while the values at a depth of 2 mm were recorded directly under the grafted skin on the surface of the muscle. This also explains the higher amounts of blood flow in deeper tissue layers.

In this study, at a depth of 2 mm the mean SO2 value significantly decreased on day 10 compared with healthy skin and remained decreased for the entire study period. The decrease in SO2 can be explained by an increase in oxygen extraction due to metabolic processes during wound healing. These include collagen synthesis as well as the formation of new vessels and their degradation during scar formation.^18,19 It is interesting that SO2 stays decreased a long time after macroscopic wound healing, due to the aforementioned processes.

In the current study, the mean SO2 at a depth of 8 mm increased compared with healthy skin over the entire study period. This may occur for 2 main reasons. First, after a tissue defect was created down to the fascia, which was then grafted, the probe measured deeper in the muscle than the probe on healthy skin, because the probe was lying directly on the skin-grafted muscle of the rats, whereas the probe on healthy skin was lying on intact skin and subcutaneous tissue. Thus, 8 mm below the intact skin was not as deep in the muscle tissue as 8 mm below the grafted muscle. Second, the deeper tissue layers receive an increased blood supply due to hyperemia caused by vasodilation and neoangiogenesis during the process of wound healing, while oxygen extraction in the muscle itself is not increased.²⁰

In the current study, the superficial blood flow at a depth of 2 mm was considerably increased on day 10 compared with baseline values of healthy tissue. Superficial blood flow at a depth of 2 mm then slowly decreased until it reached values below the baseline on day 60. Subsequently, it slightly increased again over the remainder of the study.

The blood flow in deep tissue layers (8 mm) increased over the entire measurement period, with the highest value on day 10. It remained high, with some variation, until day 40 and then slowly decreased again until it reached the baseline value on day 84.

The vast increase in blood flow on day 10 in the current study can be explained by inflammatory processes that cause vasodilatation, and therefore increased blood flow during the initial phase of wound healing (ie, the inflammatory phase).²¹ Thereafter, blood flow decreased but remained elevated compared with normal tissue. This can be explained by the transition into the proliferation phase of wound healing, during which granulation tissue is formed and increased blood flow is maintained through neoangiogenesis while vasodilatation decreases.²² The further decrease in blood flow can be explained by the wound entering the remodeling phase, which is characterized by the degradation of excess vessels and extracellular matrix through apoptosis.^23,24

Compared with the baseline of healthy tissue in the current study, rHb was increased only on day 10 at a depth of 2 mm and only on days 10 and 20 at a depth of 8 mm. After those time points, rHb in treated skin remained below the baseline value with slight variation. The faster decrease in rHb reflects a reduction in the amount of blood that is present in the capillaries. This can be explained by the initial effects of vasodilation, followed by the decrease of dilated vessels, the increase of newly formed dermal matrix, and the gradual formation of new small vessels during neoangiogenesis.^20,23

In the current study, the authors were able to monitor the complex changes in microcirculation in the course of wound healing after skin grafting. To the authors’ knowledge, no other studies have monitored microcirculation during healing of split-thickness skin grafts; however, the findings of this study correlate with those of the above-mentioned studies on the complex processes that occur during the different stages of wound healing regarding vasodilation, neoangiogenesis, and the degradation of excess vessels.

This study of the changes in microcirculation is particularly useful because the authors believe that the early stages of wound healing are crucial to outcomes such as scar quality. Thus, monitoring microcirculation may be a useful noninvasive means of identifying alterations in the healing process that can be applied to discover, for example, how and if wound dressings or certain drugs improve the healing process.

One limitation of the current study is that microcirculatory changes during the first few days following skin grafting were not monitored; this decision was made to avoid jeopardizing the vulnerable, freshly grafted skin in the wound bed. However, it would have been interesting to further investigate microcirculatory adaptions to the wounding and skin grafting of the tissue in those early days. Furthermore, only the treated tissue, but not the skin graft revascularization, was evaluated due to the 2-mm and 8-mm penetration depth of the O2C device. However, the observation period provided exceptional insights into the long-lasting healing process after skin grafting. Moreover, there was no comparison to a sham control with wounded rats without skin grafting. Another limitation is the use of a rat model, which does not exactly mimic human wound healing because of the differences in skin morphology between rats and humans. Rats are so-called loose-skinned animals, with their higher skin elasticity making wound contraction an important part of the wound healing process and decreasing time to wound healing because it is faster than reepithelialization.²⁵ In addition, rats possess a subcutaneous muscle called the panniculus carnosus that helps wound healing by contraction and collagen formation.²⁶ These differences certainly result in physiological discrepancies in the wound healing process in rats versus humans, but because these differences are reflected in the duration of wound healing rather than the microcirculatory changes themselves during wound healing, the authors of the current study believe the findings are valid for the healing process of split-thickness skin grafts in humans as well.

The microcirculatory changes noted in the current study reflect the different phases of wound healing. Long after macroscopic healing of the skin transplants was noted, alterations in microcirculation could still be detected in the wound bed because of the long-lasting changes in tissue metabolism due to the formation, conversion, and degradation of the dermal matrix and vessels during wound healing and scar formation. The authors of the current study recommend further studies performed in pigs in clinically relevant time frames (eg, after 4, 7, 14, 28, and 56 days). 

Authors: Sabrina Krauß, MD; Claudius Illg, MD; Manuel Held, Prof.; Adrien Daigeler, Prof; and Wiebke Eisler, MD

Affiliation: Eberhard Karls University of Tübingen, Tübingen, Germany

Disclosure: The authors disclose no financial or other conflicts of interest.

Correspondence: Wiebke Eisler, MD; Eberhard Karls University of Tübingen, BG Klinik Tübingen, Department of Hand, Plastic, Reconstructive and Burn Surgery, Schnarrenbergstrasse 95, Tübingen, Baden-Wuerttemberg 72076 Germany; w.v.petersen@gmx.de

Manuscript Accepted: March 21, 2024