Efficacy of Ultrasonography at the Ankle Level for Estimation of Pedal Microcirculation.

Chronic wounds due to diabetes mellitus (DM) and/or peripheral arterial disease (PAD) often occur in the pedal region peripheral to the ankle. To predict wound healing potential of limb ulcers, skin perfusion pressure (SPP) and transcutaneous oxygen tension (TcPO2) have recently become popular as the parameters that reflect skin microcirculation. On the other hand, ultrasonography for the macrocirculatory vessels has already prevailed widely as the standard vascular investigation. The skin microcirculation peripheral to the ankle probably depends on the macrocirculatory blood flow at the ankle level. Thus, this study aims to estimate whether the blood flow of the anterior tibial artery (ATA) and the posterior tibial artery, at the ankle level, reflect the values of SPP and TcPO2 on the foot. The protocol enrolled 88 patients (122 limbs) with foot ulcers due to DM and/or PAD. The statistical analysis revealed that the sum of blood flow of the ATA and the PTA (posterior tibial artery), at the ankle level, significantly correlated with SPP on the foot. The findings support the availability of conventional ultrasonographic investigation to estimate microcirculation of the foot crucial for wound healing. (This article is a translation of J Jpn Coll Angiol 2014; 54: 45-50.).

Comparison of collagen matrix treatment impregnated with platelet rich plasma vs bone marrow.

This study has reported the efficacy of an autologous bone marrow-impregnated collagen matrix experimentally and clinically. Then, it reflected that platelet rich plasma (PRP) was as good a source of growth factors as bone marrow and available in a less invasive procedure. This study aimed to compare the efficacy of a PRP-impregnated collagen matrix with that of a bone marrow-impregnated collagen matrix by quantifying wound size and capillary density using genetically diabetic db/db mice. Bone marrow cells were obtained from femurs of ddy mice. Then, a small amount of collagen matrix was immersed in bone marrow suspension. This is called a bone marrow-impregnated collagen matrix. PRP was obtained from healthy human blood and a small amount of collagen matrix was immersed in PRP. This is called a PRP-impregnated collagen matrix. A bone marrow-impregnated collagen matrix and PRP-impregnated collagen matrix were applied to excisional skin wounds on a genetically healing-impaired mouse (n = 6) and wounds were evaluated 6 days after the procedure. Wounds were divided into two groups: PRP (n = 6), in which a PRP-impregnated collagen matrix was applied; and bone marrow (n = 6), in which collagen immersed in a bone marrow suspension was applied. There was no significant difference between the PRP and bone-marrow groups in the rate of vascular density increase or wound size decrease. The present study suggested that the PRP-impregnated collagen matrix promotes repair processes at least as strongly as the bone marrow-impregnated collagen matrix. Given lower invasiveness, the PRP-impregnated collagen matrix would have advantages in clinical use.

The effect of a hydrocolloid dressing containing ceramide-2 on split-thickness wounds in a laser-induced erosion model.

OBJECTIVE: Lower levels of ceramides in the stratum corneum are considered to be an etiologic factor in the dry and barrier-disrupted skin of patients with atopic dermatitis, psoriasis vulgaris, and xeroderma pigmentosum. Topical ceramides therapy has currently been used to improve skin barrier function. However, little is known about the effect of topical ceramides on wound healing. This study investigated the effect of ceramide-2 on wound healing using a diabetic mouse model. DESIGN: Using a CO(2) laser to create standard erosions on the dorsal skin of mice, the authors developed a reproducible model of split-thickness wounds. Two wounds were placed on the back symmetrically across the spine of mice. The left-side wounds were covered with the hydrocolloid dressing containing 0.3% ceramide-2, and those on the right were covered with the control hydrocolloid dressing containing no ceramides. MAIN RESULTS: Seven days after the irradiation, controls showed a transepidermal water loss (TEWL) of 66.58 g/m(2)h on average, which was significantly higher than the mean TEWL of the ceramide-treated wounds, 46.22 g/m(2)h (P < .05, Student t test). Histological assessment also revealed ceramide-2 improved recovery of the erosion morphologically. CONCLUSION: The authors' findings suggest for the first time the possibility of the novel application of ceramide as a therapeutic agent for skin erosion.

Efficacy of a poly glycolic acid (PGA)/collagen composite nanofibre scaffold on cell migration and neovascularisation in vivo skin defect model.

Application of tissue engineering currently provides promising therapeutic options in the fields of plastic surgery and wound management. The ability of scaffold material for cell proliferation and differentiation is the key for tissue engineering. This study has developed a novel nanofibre composed of poly glycolic acid (PGA) and collagen, both of which have their own respective beneficial properties. This study aimed to estimate the in vivo efficiency of the PGA/collagen nanofibre on granulation histology and its ability to induce neovascularisation. The electrospinning technique produced the PGA/collagen nanofiber with a diameter of 500 nm and weight mixing ratio of 40%. The skin defects on the mouse model were covered with PGA/collagen or a commercially available collagen matrix (n = 9). The PGA/collagen group histologically showed significantly higher cell density and a fine microstructure with greater number of migrating cells as compared to collagen matrix. Then, both materials were applied to the microcirculatory angiogenesis model. The PGA/collagen group (n = 8) revealed significantly higher functional capillary density on days 5 and 7 after application. The findings substantiated the fact that our material had a superior ability regarding cellular migration and induction of neovascularisation compared with the elementary collagen matrix product. This better result might be attributed to the nano-size effect of fine structure and the incorporation of PGA, which has been associated with enhanced angiogenesis.

Influence of oxygen on wound healing dynamics: assessment in a novel wound mouse model under a variable oxygen environment.

INTRODUCTION: Although oxygen is essential for the wound healing process, tissue hypoxia is known to stimulate angiogenesis. To explore these inconsistent findings, we estimated the influence of the oxygen environment on wound healing with our original model. METHODS: Experiment 1 (Establishment of the model): To modify the topical oxygen tension, oxygen impermeable (polyvinylidene chloride) and permeable (polymethylpentene) membranes were applied to symmetrical excisional wounds in ddy mice (n = 6). Oxygen tension under the membrane was quantified with a device using photo-quenching technique. Experiment 2 (Influence of oxygen environment on wound healing): The wound area, granulation thickness and vascular density were analyzed under different oxygen environments (n = 24). RESULTS: Experiment 1: The permeable group maintained equivalent oxygen level to atmosphere (114.1±29.8 mmHg on day 7), while the impermeable group showed extremely low oxygen tension (5.72±2.99 mmHg on day 7). Accordingly, each group was defined as the normoxia group and the hypoxia group. Experiment 2: Percent decrease in wound size was significantly enhanced in the normoxia group (11.1±1.66% on day 7) in comparison with the hypoxia group (27.6±3.47% on day 7). The normoxia group showed significantly thicker granulation tissue than the hypoxia group (491.8±243.2 vs. 295.3±180.9 µm). Contrarily, the vascular density of the hypoxia group significantly increased on day 7 (0.046±0.025 vs. 0.011±0.008 mm(2)/mm(2)). CONCLUSIONS: Our original model successfully controlled local oxygen concentration around the wound, and the hypoxic wounds showed increased angiogenesis but with a smaller amount of granulation tissue and delayed wound closure. Enhanced neovascularization in the hypoxic group likely implies compensative response to an insufficient ambient oxygen supply.

Treatment of venous ulcers with bone marrow-impregnated collagen matrix.

Bone marrow attracted our attention as a potentially beneficial material for the treatment of wounds, because it contains multipotential progenitor cells and produces growth factors. We impregnated autologous bone marrow cells on to a collagen matrix that had been used for the treatment of chronic wounds. The bone marrow-impregnated collagen matrix was then as a biomaterial scaffold for the treatment of wounds. This study was designed with the aim of clinically evaluating the effects of bone marrow-impregnated collagen matrix on wound healing of venous ulcers. We applied the matrix in 15 patients with chronic venous ulcers, and evaluated the transcutaneous oxygen tension (TcPO(2)) and vascular density. The application of the matrix induced healthy granulation tissue. All patients were given a split-thickness skin graft on to the induced granulation tissue, and have remained free from complications for more than eight months since the treatment. The mean (SD) vascular density at the ulcer base increased after the treatment (before 0.011 (0.006) mm(2)/mm(2), after 0.064 (0.036) mm(2)/mm(2), p < 0.001). The periwound TcPO(2) values tended to increase (before 17.1 (12.7) mmHg, after 30.6 (13.4) mmHg, p < 0.001). Our results have shown the efficacy of bone marrow-impregnated collagen matrix for the treatment of intractable venous ulcers.

In vivo analysis of skin microcirculation and the role of nitric oxide during vibration.

Studies in healthy volunteers and patients with renal failure have shown that vibration, applied with a frequency of 47 Hz and a vibrational intensity of 600 mVpp, increases microcirculation of blood in the skin. This controlled, in vivo, experimental study was conducted to further evaluate the effect of vibration on skin microcirculation and to ascertain whether administration of a nitric oxide (NO) synthase inhibitor (NG-nitro-L-arginine methyl ester [L-NAME]) diminishes the effect of vibration on skin blood flow. Using a mouse microcirculatory model, 12 animals were prepared for study (six in the control and six in the experimental group). In the experimental group, vibrations were applied horizontally for 15 minutes. The control group received no vibration. Venular blood flow was measured using intravital videomicroscopy at baseline and at 0, 5, and 15 minutes after the application of vibration. Vibration significantly increased the blood flow at 5 and 15 minutes after application (P = 0.002 and P = 0.046, respectively). Differences between the control and experimental group also were statistically significant (P = 0.0017 and P = 0.046, respectively). In the second study, all animals (seven in each group) received an intraperitoneal injection of NO synthase inhibitor L-NAME before vibration application. When NO synthase inhibitor L-NAME was administered, the increase in blood flow in the vibration group was minimal after 5 and 10 minutes, and nonexistent after 15 minutes. No significant differences between the control and experimental group were observed. Because NO synthase inhibitor L-NAME inhibits NO production in vivo, these findings imply the involvement of NO in the observed blood flow increase during vibration. Future clinical trials to establish evidence as to the beneficial effects of vibration are warranted.

Determinants of wound healing in bone marrow-impregnated collagen matrix treatment: impact of microcirculatory response to surgical debridement.

Although previous reports have suggested the efficacy of autologous bone marrow-impregnated collagen matrix experimentally and clinically, we occasionally encounter difficult wounds that fail to respond to the treatment. The current study retrospectively investigated the factors that affect clinical outcomes based on the hypothesis that periwound microcirculation may play a significant role. Fifty-three patients with chronic wounds received surgical debridement, followed by application of an autologous bone marrow-impregnated collagen matrix. The periwound transcutaneous oxygen tension (TcPO(2)) was evaluated (n=39). The patients were retrospectively divided into successful and unsuccessful subgroups. Successful treatment was defined as wound closure by spontaneous healing or skin graft. The TcPO(2) of the unsuccessful subgroup significantly decreased after debridement while that of the successful subgroup increased. Among various parameters, the TcPO(2) at 4 days after debridement showed the strongest association with the success of the treatment. As reference data, we collected the information of the patients (n=22) who received standard wound care, and they showed the same trend wherein the TcPO(2) of the unsuccessful subgroup markedly decreased after debridement. Reactivity of the wound microcirculation to increased wound perfusion in response to the surgical debridement might be a key determinant for successful wound healing.

A technique to visualize wound bed microcirculation and the acute effect of negative pressure.

Although previous studies have proved that subatmospheric pressure in vacuum assisted closure therapy increased blood flow at the wound edge, no reports have presented data on blood flow in the wound bed. This study examined a technique that visualized microcirculation of the wound bed and estimated the acute effect of negative pressure. The superficial stratum of the mouse gluteal skin was microsurgically excised preserving the subdermal vascular plexus. The preserved vessels were visualized as the wound bed microcirculation using an intravital microscope-video-computer system. Three levels of negative pressure (-125 mmHg [n=12], -500 mmHg [n=12], 0 mmHg [n=8]) were applied to the wound. Our experimental model successfully and quantitatively visualized wound bed microcirculation under negative pressure application. A negative pressure of -125 mmHg significantly increased wound bed blood flow immediately after pressure application, maintained for 1 minute after pressure release, whereas in the -500 mmHg group blood flow decreased with time and reached a statistically significant level 5 minutes after pressure application. These findings reinforce the hypothesis that enhanced blood flow in the wound bed as well as at the wound edge may contribute to the beneficial effects of certain levels of negative pressure therapy for wound perfusion.

Oxygen consumption of keloids and hypertrophic scars.

The oxygen consumption of keloids and hypertrophic scars has never been quantitatively presented, although abnormal metabolic conditions must be associated with their pathophysiology. We invented an original measurement system equipped with a Clark oxygen electrode for ex vivo samples. The measurement of a mouse wound-healing model revealed immature repairing tissues consumed more oxygen than mature tissues. This finding is in accord with the current thinking and supported the validity of our measurement system. The analysis of fresh human samples clearly demonstrated the high oxygen consumption rate of keloid hypertrophic scars and the comparatively low consumption of mature scars. A high oxygen consuming potential, as well as insufficient oxygen diffusion, may possibly contribute to the pathophysiology of keloids and hypertrophic scars.

AlphaV beta3 (alphavbeta3) integrin inhibition reduces leukocyte-endothelium interaction in a pressure-induced reperfusion model.

The leukocyte-endothelium interaction is known to contribute to reperfusion injury, which is considered to participate in the pathophysiology of pressure ulcers, and integrin alphaV beta3 (alphavbeta3) has been shown to mediate the processes of cellular adhesion in various types of cells. This study aims to clarify leukocyte behavior in our original microcirculatory pressure-induced reperfusion model, which can visualize the microcirculation in vivo. We also estimated the effect of alphavbeta3 integrin inhibition on the reduction of the leukocyte-endothelium interaction. Mice with dorsal skinfold chambers were divided into three groups: the baseline group (n=6), in which animals received no compression; the compression-reperfusion group (n=6), in which animals underwent 2-hour compression of the dorsal skin, followed by release, and the inhibitor-treated group (n=7), in which an alphavbeta3 inhibitor, CP4715, was administered in addition to the compression-release procedure. Staining with rhodamine 6G quantitatively visualized leukocyte behavior under the intravital fluorescent microscope. Compression-reperfusion induced a significant increase in rolling, sticking, and extravasation of the leukocytes. Treatment with the inhibitor strikingly reduced leukocyte sticking and extravasation. The present experiment has provided evidence that alphavbeta3 inhibition reduces leukocyte-endothelium interaction in our original pressure-induced reperfusion model.

Effect of vibration on skin blood flow in an in vivo microcirculatory model.

The effect of vibration on skin microcirculation was studied to investigate the possibility of clinical use of vibration to prevent and treat pressure ulcers. Vibrations at a vibrational intensity of 600, 800, or 1,000 mVpp with a fixed frequency of 47 Hz were applied horizontally to the ear of male hairless mice (n = 6 for each group) under inhalation anesthesia. The control group (n = 6) received no vibrations. Venular blood flow was measured by an intravital videomicroscope at the baseline and at 0, 5, and 15 min after the application of vibrations. A significant increase was observed in the 600 mVpp group 5 and 15 min after vibration in comparison to the control group (P = 0.002 and P = 0.046, respectively). We also detected increased blood flow in the 800 mVpp group (P = 0.028) and the 1,000 mVpp group (P = 0.012) 5 min after vibration; however, these increases attenuated after 15 min. These results indicate that direct skin vibration at a frequency of 47 Hz improves skin blood flow. The present study gives further support to the role of vibration on a short-term increase in skin blood flow.

Analysis of ischemia-reperfusion injury in a microcirculatory model of pressure ulcers.

The aim of this study was to establish a pressure ulcer model that visualizes the microcirculation, and to examine the participation of ischemia-reperfusion injury in the pathophysiology of pressure ulcers. An original system composed of a new skin fold chamber and compression device allowed loading quantitative vertical stress to the skin. An intravital microscopic technique enabled direct visualization of the microcirculation in the physiological condition and in response to pressure application. To estimate the effect of ischemia-reperfusion injury, animals were divided into two groups: the compression-release group (n = 8), in which the animals received four cycles of compression-release which consisted of 2 hours of compression followed by 1 hour of pressure release; and the compression alone group (n = 8) in which the animals underwent continuous compression for 8 hours. Functional capillary density was quantified before the compression procedure and on day 1 (35 hours) after the first evaluation. The cyclic compression-release procedure significantly decreased functional capillary density as compared to continuous compression, indicating that in our experimental setting repetition of ischemia-reperfusion cycle more severely damaged the microcirculation than single prolonged ischemic insult. This finding supports the significant contribution of ischemia-reperfusion injury to the pathophysiology of pressure ulcers at the level of dynamic in vivo microcirculation.

Bone marrow cell implantation improves flap viability after ischemia-reperfusion injury.

This study attempted to clarify the effects of therapeutic neovascularization by bone marrow cells for salvaging flaps after ischemia-reperfusion injury. Bone marrow mononuclear cell layer (endothelial progenitor cell-enriched fraction) was isolated from the mouse femur and tibia. Symmetrical double flaps were elevated in mice. Each flap topically received phosphate buffered saline (PBS) or bone marrow cells in PBS. Flaps were subjected to 6-hour ischemia and subsequent reperfusion. On the seventh postoperative day, the flap survival area was measured (n = 27). The mean survival area of bone marrow cells-transplanted flaps was 66.3 +/- 18.0%, whereas control flaps showed a survival area of 49.7 +/- 22.2%. The difference was highly significant (P = 0.000209). Histologic examination revealed the average vascular density of bone marrow cells-transplanted flaps had significantly increased. The present study proved bone marrow cells acted with significant efficacy in promoting the survival of ischemia-reperfusion-mediated damaged tissue.

Regenerative surgery for sacral pressure ulcers using collagen matrix substitute dermis (artificial dermis).

Collagen matrix substitute dermis, or artificial dermis, has recently been developed to induce angiogenesis and fibroplasia in deep, poorly vascularized tissue defects, and its use is desirable as a means of achieving effective treatment with less invasion. However, it is difficult to apply collagen matrix to pressure ulcers, because they are usually accompanied by infection with discharge of excessive amounts of exudate or pus and generally exposed to external forces that prevent graft fixation. To overcome these difficulties, the authors demonstrated a novel procedure to exclude unfavorable exudate and external forces. The procedure resulted in successful fixation of the artificial dermis and induction of regeneration in poorly vascularized defects in every case. The histologic findings provided supporting evidence that collagen matrix acts as a scaffold for reconstruction of nearly normal vascular structures coursing perpendicularly in the upper layer of normal dermis.

In vivo model for visualizing flap microcirculation of ischemia-reperfusion.

Since ischemia-reperfusion injury continues to be a major problem in reconstructive microsurgery, improvement of experimental models is still desirable. We developed a model that allows direct visualization of flap microcirculation in mice by intravital microscopic techniques. A newly designed skinfold chamber was installed on the dorsum of mice, and microcirculation was inspected with an intravital microscope. An island flap, nourished by the deep circumflex iliac arteries, was elevated after implantation of the chamber, allowing visualization of the microcirculation in the island flap. The island flap was exposed to global ischemia by clamping the pedicles, and the clamps were then released to allow reperfusion. Various microcirculatory responses induced by ischemia-reperfusion were visualized. This model accurately simulated the clinical situation in reconstructive surgery and successfully realized chronic visualization of the flap microcirculation in vivo.