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The Laryngoscope

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Enhancement of Ischemic Wound Healing by Inducement of Local Angiogenesis

Hannah S. Milch, BA; Shai Y. Schubert, PhD; Stephen Hammond, MD; Jeffrey H. Spiegel, MD

Objective: To determine if monocytes activated toward an angiogenic phenotype can be used to improve ischemic tissue healing in a rat skin flap model. Study Design: Prospective experimental study on Wistar rats. Methods: A caudally based 9 3 cm dorsal skin/panniculus carnosus flap was raised in 15 rats. The animals were divided into three groups: the monocyte group (N 5) received subcutaneous topical application of 0.10.2 cc of i-MonogridTM, a collagen gel containing M2 angiogenic monocytes; control group 1 (N 5) received application of cell-free collagen; and control group 2 (N 5) received no treatment. Skin flaps were stapled in place and observed for wound ischemia and necrosis of the skin flap. One week postoperatively, skin and underlying muscle were harvested for histologic analyses. Results: No macroscopic differences in wound healing or microscopic differences in skin viability were observed. However, the monocyte group showed significantly greater vascular improvement than C1 (P .047, v 3.96), and a trend toward greater vascular improvement than C2 (P .103, v 2.67). Conclusions: Delivery of activated pro-angiogenic monocytes to an ischemic skin flap tended to improve histologic evidence of vascularity without corresponding microscopic or gross evidence of improved flap survival. These results are encouraging regarding the use of monocytes as a potential method of improving vascularization of ischemic tissue.

Key Words: Skin flap necrosis, monocytes, angiogenesis. Level of Evidence: 5 Laryngoscope, 120:17441748, 2010

INTRODUCTION
The objective of this study was to evaluate the efficacy of angiogenic monocytes embedded in a biodegradable matrix in the treatment of ischemic wounds. Monocytes have the potential to differentiate into different functional phenotypes based on their microenvironment.1,2 The M2 alternative pathway of monocyte differentiation refers to the pro-angiogenic phenotype of monocytes.3,4 This study examined the potential of monocytes directed toward their angiogenic (M2) phenotype to enhance ischemic wound healing by increasing angiogenesis. Local delivery of progenitor cells to ischemic wounds has been demonstrated to enhance wound healing.57 For example, McFarlin et al.8 showed that systemic administration of bone marrow-derived mesenchymal stromal cells improved wound healing in rats. However, the availability of stem cells for therapeutic use is limited, and the use of pro-angiogenic monocytes could prove to be a viable alternative. Monocytes are readily available by separation from peripheral blood and can be easily obtained in large numbers from patients. Monocytes may provide a source for therapeutic angiogenic cells, and have the ability improve skin flap surgery outcomes and the treatment of ischemic wounds. The therapeutic applications are many, as increased angiogenesis can ideally make healing more rapid and robust, particularly in environments with potentially compromised healing. These would include in people who smoke, those with prior radiation or other harmful treatments, individuals with comorbidities such as diabetes mellitus, and those in whom skin flap design was suboptimal. Evaluation of the potential of angiogenic monocytes embedded in 3D matrices for the treatment of ischemic wound healing was carried out with two specific goals: 1) to determine the ability of collagen gel embedded with M2 angiogenic monocytes to reduce ischemic injury (necrosis) in single-pedicle dorsal skin flap injury in rats; Milch et al.: Angiogenesis in Ischemic Skin Wound

From the Boston University School of Medicine (H.S.M.), Boston, Massachusetts, U.S.A.; Moma Therapeutics ( S . Y. S .), Brighton, Massachusetts, U.S.A.; Department of Pathology and Laboratory Medicine (S.H.), Boston Medical Center, Boston, Massachusetts, U.S.A.; Department of OtolaryngologyHead and Neck Surgery (J.H.S.), Boston University School of Medicine, Boston, Massachusetts, U.S.A. Editors Note: This Manuscript was accepted for publication April 26, 2010. Financial disclosure information: Shai Y. Schubert, PhD, is President of Moma Therapeutics, Brighton, MA; Jeffrey H. Spiegel, MD, is on the Advisory Board of Moma Therapeutics, Brighton, MA. The authors declare no conflicts of interest. This was a TRIO Section Meeting Oral Presentation. Send correspondence to Dr. Jeffrey H. Spiegel, Boston University School of Medicine, 830 Harrison Avenue, Suite 1400, Boston, MA 02118. E-mail: Jeffrey.Spiegel@bmc.org DOI: 10.1002/lary.21068

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Fig. 1. Gross images of skin flaps 7 days postoperation. Column 1 contains images of animals in the monocyte group, who received collagen matrix embedded with angiogenic monocytes. Column 2 contains images of control group 1 (C1), animals who received cell-free collagen application. Column 3 contains images of control group 2 (C2), animals who received no application. *Note the image of skin flap from animal in column 3, row 5, was taken from day 8 postoperation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

and 2) to measure the effect of collagen gel embedded with M2 angiogenic monocytes on angiogenesis.

METHODS
In this study, monocytes polarized toward the M2 angiogenic phenotype were embedded in i-MonogridTM, a biodegradable

matrix of collagen and polycaprolactone particles (PCL) designed to support monocytes in their angiogenic phenotype (Moma Therapeutics, Brighton, MA). The monocyte-embedded i-MonogridTM gel was then applied locally at the site of an ischemic wound using an animal model of female Wistar rats. It was hypothesized that autologous monocytes directed toward their angiogenic (M2) phenotype would enhance ischemic wound healing by increasing angiogenesis.

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Fig. 2. Viability of skin klap. (a) Full thickness epidermis with underlying dermis showing functional adnexal structures (>50% viability); (b) necrotic epidermis and dermis with adnexal dropout (<10% viability). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

This study was approved after full review by the Institutional IACUC committee at Boston University School of Medicine. i-MonogridTM was provided by Moma Therapeutics. Monocytes were separated from rat blood obtained by homologous collection. Following gradient centrifugation of blood [50% in phosphate-buffered saline (PBS)] on Histopaque-1077, monocytes were separated from the mononuclear cell fraction employing a negative isolation method based on the depletion of nonmonocytes by their binding to antibody coated magnetic beads (Miltenyi Biotec, Auburn, CA). The separation results in high purity (>90%) of untouched monocytes. A total of 5 cc of blood were taken from a donor rat approximately 250 g in size. Monocytes were incubated in 100 nM adenosine in PBS, 2.5 mM EDTA for 1 hour prior to addition of collagen. Animals (N 15) were anesthetized by intraperitoneal injection of xylazine and ketamine (4080 mg/kg ketamine and 510 mg/kg xylazine), with perioperative analgesia with subcutaneous buprenorphine at the time of surgery and doses of 0.05 mg/kg subcutaneously every 12 hours for 2 days postoperatively. A caudally based 9 3 cm dorsal skin/panniculus carnosus flap was raised with the two constant sacral axial vessels systematically cut. This skin flap design is a modification of the single pedicle dorsal skin flap previously published as a model for ischemic skin flap healing.9 The caudal border of the flap was marked at 1 cm below the posterior iliac crests. The animals were divided into three groups: a monocyte treated group and two control groups. In the distal end of the skin flap, the monocyte group (N 5) received subcutaneous topical application of 0.10.2 cc of i-MonogridTM, a collagen gel containing M2 angiogenic monocytes. Approximately 200 400,000 monocytes were delivered to each animal in the treatment group. Control group 1 (C1, N 5) received subcutaneous topical application of cell free collagen. Control group 2 (C2, N 5) received no applications. All collagen and monocyte applications occurred while the animals were sedated, shortly after operation and before wound closure. The use of type 1 human collagen as the carrier for the monocytes was used to minimize the risk of adverse reactions. The skin flaps of all animals were then repositioned and stapled in place. After the surgical procedure, the animals were returned to individual cages and received food and water ad libitum. Evalu-

ation of wound healing was monitored using analysis of images of the injury taken every 24 hours during the course of the study. At day 7 postoperation, the skin flaps were harvested for histologic analyses. For analysis, the flap was divided into proximal and distal portions. The proximal portion was the area of the flap closer to the scapula and was expected to behave similar to normal tissue. In contrast, severe ischemia and necrosis were expected in the distal portion of the flap. To evaluate wound angiogenesis, vascularity in the distal sections of the skin flap was measured at day 7 postoperation. Wound healing was characterized by evaluation of skin flap recovery using both visual parameters and histology of the wound at day 7 postoperation. To evaluate wound angiogenesis, a pathologistblinded to animal groupqualitatively assessed the density of neocapillarization within each skin flap. Four representative tissue sections were evaluated from each animal, such that a total of 20 tissue sections were assessed in each group of five animals. Vascularization was measured on a scale of 0 to 100, where the least vascularized graft was defined as 0, and the most vascularized graft was defined as 100. Grafts were given a score based on the level of vascularization within this scale. Improved vascularity was defined as greater than 50% vascular density of the skin flap relative to the degree of vascularity in all tissue samples. Chi-squared analyses were used to determine differences in vascularity among groups.

RESULTS Macroscopic
Necrosis was observed in all animals by 35 days following the operation, marked by hardening of the skin, darkening, and loss of hair. At day 7, necrotic tissue encompassed one-quarter to one-half of the distal portion of all skin flaps (Fig. 1). The extent of necrosis among individual animals was not diminished within any one particular group. These results suggest that ischemia led to poor wound healing in all rats, regardless of treatment with pro-angiogenic monocytes. Milch et al.: Angiogenesis in Ischemic Skin Wound

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Fig. 3. Vascularity of skin flap. (a) Neovascularization: new capillary growth within loose myxoid stroma typical of angiogenesis (>50% vascular density); (b) absence of angiogenesis illustrated by lack of new vessels within a myxoid stroma (<10% vascular density). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Histology
Histologic evaluation of the skin flap focused on two distinct parameters: skin viability and skin vascularity. Viability of the skin was evaluated based on the presence of full thickness of the epidermis, an underlying dermis, and intact adnexal structures (Fig. 2a). Nonviable skin showed evidence of a necrotic epidermis and dermis, and no functional adnexal structures (Fig. 2b). Vascularity of the skin was evaluated based on the degree of neocapillarization of the skin and subcutis (Fig. 3). Tissue vascularity did not appear to depend on skin viability, that is, a skin section with minimal intact dermis and epidermis often corresponded with an abundance of underying granulation tissue and new blood vessel growth. Conversely, an intact dermis and epidermis often had minimal underlying blood growth. The results were categorized into four percentage ranges <10%, 10% to 20%, 20% to 50%, >50%for both extent of vascularity and extent of viability in representative tissue sections (Fig. 4).

significant difference in vascular improvement between the two control groups (P .705, v 0.143).

DISCUSSION
The goal of the monocyte treatment was to increase neovascularization of the skin flap in order to reduce ischemia and improve wound healing. We hypothesized that application of a biodegradable collagen matrix containing pro-angiogenic monocytes would improve vascularization of the skin, lead to an increase in skin viability, and reduce necrosis. A trend toward improved

Viability
No group differences were observed in skin flap viability.

Vascularity
A trend toward improved vascularitydefined as greater than 50% vascular densitywas observed in the monocyte group. The monocyte group exhibited more than double the amount of vascular improvement as the two control groups (Fig. 4). The monocyte group showed significantly greater vascular improvement than C1 (P .047, v 3.96), and a trend toward greater vascular improvement than C2, although this comparison was not statistically significant (P .103, v 2.67). A three-way comparison also showed a trend toward differences in vascular improvement (P .092), although this difference was not statistically significant. There was no Laryngoscope 120: September 2010

Fig. 4. Distribution of vascular density among groups. The dorsal portion of each skin flap was divided into four equal-sized segments for histological analysis, resulting in a total of 20 skin flaps examined in each group of five animals. Green indicates number of tissue sections with greater than 50% vascular density; yellow indicates number of tissue sections with 20%50% vascular density; red indicates number of tissue sections with 10%20% vascular density; and blue indicates number of tissue sections with less than 10% vascular density. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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vascularity was seen in the treatment group compared to the two control groups. However, this improved vascularity was not associated with improved flap survival, and no differences in skin viability or degree of necrosis were observed among the groups after 7 days of wound healing. Our results suggest that pro-angiogenic monocytes may result in improved vascularization of the skin flap, or at least of the skip flap environment, but that this neovascularization was not sufficient to improve wound healing. Both the quantity and the quality of the monocyte therapy may explain this phenomenon. Homologous monocytes from a single rat donorrather than autologous monocyteswere embedded in the matrix applied to all rats in the monocyte group. Autologous monocyte therapy may have been more effective in improving flap survival, but due to the animal size, was not a viable test technique. Homologous monocytes also introduce the risk of immune destruction of the tissue, which in turn could further hinder the wound-healing process. In clinical practice, it would be desirable to use autologous monoctye therapy. In addition, due to animal size, a relatively limited number of monocytesapproximately 200400,000 per animalwere obtained and embedded in the collagen matrix. A greater number of autologous monocytes may have further acted to create enough new blood vessel growth to enable increased skin flap survival. The experimental methods used to assess the monocyte therapy had several potential limitations. Additional changes in duration of healing and route of treatment administration may have further enhanced the results. The duration of wound healing observation7 days following the operationmay have been too short to see gross differences in wound healing. A longer duration for wound healing may have further increased neovascularization and yielded an observable improvement in skin viability. The method of application may have been insufficient to deliver the monocyte therapy effectively. Treatment was limited to the underlying distal portion of the skin flap. Application of i-MonogridTM under the entire flap area may be required in order to allow better blood flow toward the distal end of the flap. Topical application may be a self-limiting technique for therapeutic angiogenesis as it forms a physical barrier between the flap and the underlining tissue. Injection to the flap may be a better mode of delivery in order to achieve angiogenesis within the flap. Additional trials will permit elucidation of the best application technique. In addition, the complexity of a composite ischemic skin flap model may have been too challenging to be treated with topical monocyte therapy alone. A more simple ischemic skin flap modelof smaller size or a different locationmay have been a better approach to test the efficacy of the treatment. It is also possible that contamination hindered the wound healing process. The

surgical operation was completed in a clean, but not sterile, environment, and infection may have adversely affected the monocyte therapy. Nonetheless, it is provocative to observe such a degree of increased wound vascularity in the treatment group. The activity of the i-MonogridTM appears promising for improvement of the wound-healing environment. Finally, it is important to address the potential oncogenic effect of increasing angiogenesis, especially in patients suffering from postradiation therapy recurrences. The potential for adverse outcomes following cancer treatment by increasing angiogenesis at a resection site remains unknown. However, general practice remains to maximize the ability for complex wounds to heal. Therefore, monocyte therapy may prove to be a useful adjunct, even in patients with a history of local malignancy.

CONCLUSIONS
Delivery of activated pro-angiogenic monocytes to an ischemic skin flap tended to improve histologic evidence of vascularity without corresponding microscopic or gross evidence of improved flap survival. These results are encouraging regarding the use of monocytes as a potential method of improving vascularization of ischemic tissue. A greater number of monocytes in each dose, or injection rather than topical application, may yield improved results in other applications.

BIBLIOGRAPHY
1. Martinez FO, Gordon S, Locati M, Mantovani A. Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: new molecules and patterns of gene expression. J Immunol 2006;177:73037311. 2. Mantovani A, Sica A, Locati M. New vistas on macrophage differentiation and activation. Eur J Immunol 2007;37: 1416. 3. Crowther M, Brown NJ, Bishop ET, Lewis CE. Microenvironmental influence on macrophage regulation of angiogenesis in wounds and malignant tumors. J Leukoc Biol 2001; 70:478490. 4. Mantovani A, Sica A, Locati M. Macrophage polarization comes of age. Immunity 2005;23:344346. 5. Cha J, Falanga V. Stem cells in cutaneous wound healing. Clin Dermatol 2007;25:7378. 6. Velazquez OC. Angiogenesis and vasculogenesis: inducing the growth of new blood vessels and wound healing by stimulation of bone marrow-derived progenitor cell mobilization and homing. J Vasc Surg 2007;45(Suppl A): A39A47. 7. Chan RK, Garfein E, Gigante PR, Liu P, Agha RA, Mulligan R, Orgill DP. Side population hematopoietic stem cells promote wound healing in diabetic mice. Plast Reconstr Surg 2007;120:407411; discussion 412403. 8. McFarlin K, Gao X, Liu YB, et al. Bone marrow-derived mesenchymal stromal cells accelerate wound healing in the rat. Wound Repair Regen 2006;14:471478. 9. McFarlane RM, DeYoung G, Henry RA. The design of a pedicle flap in the rat to study necrosis and its prevention. Plast Reconstr Surg 1965;35:177182.

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