enhancement of ischemic wound healing by inducement of local angiogenesis

The LaryngoscopeVC 2010 The American Laryngological,Rhinological and Otological Society, Inc.

Enhancement of Ischemic Wound Healing byInducement of Local Angiogenesis

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

Objective: To determine if monocytes activatedtoward an angiogenic phenotype can be used toimprove ischemic tissue healing in a rat skin flapmodel.

Study Design: Prospective experimental studyon Wistar rats.

Methods: A caudally based 9 � 3 cm dorsalskin/panniculus carnosus flap was raised in 15 rats.The animals were divided into three groups: themonocyte group (N ¼ 5) received subcutaneous topicalapplication of 0.1–0.2 cc of i-MonogridTM, a collagengel containing M2 angiogenic monocytes; controlgroup 1 (N ¼ 5) received application of cell-free colla-gen; and control group 2 (N ¼ 5) received no treat-ment. Skin flaps were stapled in place and observedfor wound ischemia and necrosis of the skin flap. Oneweek postoperatively, skin and underlying musclewere harvested for histologic analyses.

Results: No macroscopic differences in woundhealing or microscopic differences in skin viabilitywere observed. However, the monocyte group showedsignificantly greater vascular improvement than C1(P ¼ .047, v ¼ 3.96), and a trend toward greater vas-cular improvement than C2 (P ¼ .103, v ¼ 2.67).

Conclusions: Delivery of activated pro-angio-genic monocytes to an ischemic skin flap tended toimprove histologic evidence of vascularity withoutcorresponding microscopic or gross evidence ofimproved flap survival. These results are encouragingregarding the use of monocytes as a potential methodof improving vascularization of ischemic tissue.

Key Words: Skin flap necrosis, monocytes,angiogenesis.

Level of Evidence: 5Laryngoscope, 120:1744–1748, 2010

INTRODUCTIONThe objective of this study was to evaluate the effi-

cacy of angiogenic monocytes embedded in abiodegradable matrix in the treatment of ischemicwounds. Monocytes have the potential to differentiateinto different functional phenotypes based on theirmicroenvironment.1,2 The M2 alternative pathway ofmonocyte differentiation refers to the pro-angiogenicphenotype of monocytes.3,4 This study examined thepotential of monocytes directed toward their angiogenic(M2) phenotype to enhance ischemic wound healing byincreasing angiogenesis.

Local delivery of progenitor cells to ischemicwounds has been demonstrated to enhance wound heal-ing.5–7 For example, McFarlin et al.8 showed thatsystemic administration of bone marrow-derived mesen-chymal stromal cells improved wound healing in rats.However, the availability of stem cells for therapeuticuse is limited, and the use of pro-angiogenic monocytescould prove to be a viable alternative. Monocytes arereadily available by separation from peripheral bloodand can be easily obtained in large numbers frompatients. Monocytes may provide a source for therapeu-tic angiogenic cells, and have the ability improve skinflap surgery outcomes and the treatment of ischemicwounds. The therapeutic applications are many, asincreased angiogenesis can ideally make healing morerapid and robust, particularly in environments withpotentially compromised healing. These would include inpeople who smoke, those with prior radiation or otherharmful treatments, individuals with comorbidities suchas diabetes mellitus, and those in whom skin flap designwas suboptimal.

Evaluation of the potential of angiogenic monocytesembedded in 3D matrices for the treatment of ischemicwound healing was carried out with two specific goals: 1)to determine the ability of collagen gel embedded withM2 angiogenic monocytes to reduce ischemic injury (ne-crosis) in single-pedicle dorsal skin flap injury in rats;

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 LaboratoryMedicine (S.H.), Boston Medical Center, Boston, Massachusetts, U.S.A.;Department of Otolaryngology–Head and Neck Surgery (J.H.S.), BostonUniversity School of Medicine, Boston, Massachusetts, U.S.A.

Editor’s Note: This Manuscript was accepted for publication April26, 2010.

Financial disclosure information: Shai Y. Schubert, PhD, is Presi-dent of Moma Therapeutics, Brighton, MA; Jeffrey H. Spiegel, MD, is onthe 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 UniversitySchool of Medicine, 830 Harrison Avenue, Suite 1400, Boston, MA 02118.E-mail: [email protected]

DOI: 10.1002/lary.21068

Laryngoscope 120: September 2010 Milch et al.: Angiogenesis in Ischemic Skin Wound

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

METHODSIn 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-embeddedi-MonogridTM gel was then applied locally at the site of anischemic wound using an animal model of female Wistar rats. Itwas hypothesized that autologous monocytes directed towardtheir angiogenic (M2) phenotype would enhance ischemicwound healing by increasing angiogenesis.

Fig. 1. Gross images of skin flaps 7 days postoperation. Column 1 contains images of animals in the monocyte group, who received collagen ma-trix embedded with angiogenic monocytes. Column 2 contains images of control group 1 (C1), animals who received cell-free collagen applica-tion. Column 3 contains images of control group 2 (C2), animals who received no application. *Note the image of skin flap from animal in column3, row 5, was taken from day 8 postoperation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]


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This study was approved after full review by the Institu-tional IACUC committee at Boston University School ofMedicine. i-MonogridTM was provided by Moma Therapeutics.Monocytes were separated from rat blood obtained by homolo-gous collection. Following gradient centrifugation of blood [50%in phosphate-buffered saline (PBS)] on Histopaque-1077, mono-cytes were separated from the mononuclear cell fractionemploying a negative isolation method based on the depletion ofnonmonocytes by their binding to antibody coated magneticbeads (Miltenyi Biotec, Auburn, CA).

The separation results in high purity (>90%) of untouchedmonocytes. A total of 5 cc of blood were taken from a donor ratapproximately 250 g in size. Monocytes were incubated in 100nM adenosine in PBS, 2.5 mM EDTA for 1 hour prior to addi-tion of collagen.

Animals (N ¼ 15) were anesthetized by intraperitonealinjection of xylazine and ketamine (40–80 mg/kg ketamine and5–10 mg/kg xylazine), with perioperative analgesia with subcu-taneous buprenorphine at the time of surgery and doses of 0.05mg/kg subcutaneously every 12 hours for 2 days postoperatively.A caudally based 9 � 3 cm dorsal skin/panniculus carnosus flapwas raised with the two constant sacral axial vessels systemati-cally cut. This skin flap design is a modification of the singlepedicle dorsal skin flap previously published as a model for is-chemic skin flap healing.9 The caudal border of the flap wasmarked at 1 cm below the posterior iliac crests.

The animals were divided into three groups: a monocytetreated group and two control groups. In the distal end of theskin flap, the monocyte group (N ¼ 5) received subcutaneoustopical application of 0.1–0.2 cc of i-MonogridTM, a collagen gelcontaining M2 angiogenic monocytes. Approximately 200–400,000 monocytes were delivered to each animal in the treat-ment group. Control group 1 (C1, N ¼ 5) received subcutaneoustopical application of cell free collagen. Control group 2 (C2, N¼ 5) received no applications. All collagen and monocyte appli-cations occurred while the animals were sedated, shortly afteroperation and before wound closure. The use of type 1 humancollagen as the carrier for the monocytes was used to minimizethe risk of adverse reactions. The skin flaps of all animals werethen repositioned and stapled in place.

After the surgical procedure, the animals were returned toindividual cages and received food and water ad libitum. Evalu-

ation of wound healing was monitored using analysis of imagesof the injury taken every 24 hours during the course of thestudy.

At day 7 postoperation, the skin flaps were harvested forhistologic analyses. For analysis, the flap was divided into prox-imal and distal portions. The proximal portion was the area ofthe flap closer to the scapula and was expected to behave simi-lar to normal tissue. In contrast, severe ischemia and necrosiswere expected in the distal portion of the flap. To evaluatewound angiogenesis, vascularity in the distal sections of theskin flap was measured at day 7 postoperation. Wound healingwas characterized by evaluation of skin flap recovery usingboth visual parameters and histology of the wound at day 7postoperation.

To evaluate wound angiogenesis, a pathologist—blinded toanimal group—qualitatively assessed the density of neocapilla-rization within each skin flap. Four representative tissuesections were evaluated from each animal, such that a total of20 tissue sections were assessed in each group of five animals.Vascularization was measured on a scale of 0 to 100, where theleast vascularized graft was defined as 0, and the most vascu-larized graft was defined as 100. Grafts were given a scorebased on the level of vascularization within this scale. Improvedvascularity was defined as greater than 50% vascular density ofthe skin flap relative to the degree of vascularity in all tissuesamples. Chi-squared analyses were used to determine differen-ces in vascularity among groups.

RESULTS

MacroscopicNecrosis was observed in all animals by 3–5 days

following the operation, marked by hardening of theskin, darkening, and loss of hair. At day 7, necrotic tis-sue encompassed one-quarter to one-half of the distalportion of all skin flaps (Fig. 1). The extent of necrosisamong individual animals was not diminished withinany one particular group. These results suggest that is-chemia led to poor wound healing in all rats, regardlessof treatment with pro-angiogenic monocytes.

Fig. 2. Viability of skin klap. (a) Fullthickness epidermis with underlyingdermis showing functional adnexalstructures (>50% viability); (b) ne-crotic epidermis and dermis withadnexal ‘‘dropout’’ (<10% viability).[Color figure can be viewed in theonline issue, which is available atwileyonlinelibrary.com.]


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HistologyHistologic evaluation of the skin flap focused on two

distinct parameters: skin viability and skin vascularity.Viability of the skin was evaluated based on the pres-ence of full thickness of the epidermis, an underlyingdermis, and intact adnexal structures (Fig. 2a). Nonvi-able skin showed evidence of a necrotic epidermis anddermis, and no functional adnexal structures (Fig. 2b).

Vascularity of the skin was evaluated based on thedegree of neocapillarization of the skin and subcutis(Fig. 3). Tissue vascularity did not appear to depend onskin viability, that is, a skin section with minimal intactdermis and epidermis often corresponded with an abun-dance of underying granulation tissue and new bloodvessel growth. Conversely, an intact dermis and epider-mis often had minimal underlying blood growth. Theresults were categorized into four percentage ranges—<10%, 10% to 20%, 20% to 50%, >50%—for both extentof vascularity and extent of viability in representativetissue sections (Fig. 4).

ViabilityNo group differences were observed in skin flap

viability.

VascularityA trend toward improved vascularity—defined as

greater than 50% vascular density—was observed in themonocyte group. The monocyte group exhibited morethan double the amount of vascular improvement as thetwo control groups (Fig. 4). The monocyte group showedsignificantly greater vascular improvement than C1(P ¼ .047, v ¼ 3.96), and a trend toward greater vascularimprovement than C2, although this comparison was notstatistically significant (P ¼ .103, v ¼ 2.67). A three-waycomparison also showed a trend toward differences invascular improvement (P ¼ .092), although this differ-ence was not statistically significant. There was no

significant difference in vascular improvement betweenthe two control groups (P ¼ .705, v ¼ 0.143).

DISCUSSIONThe goal of the monocyte treatment was to increase

neovascularization of the skin flap in order to reduce is-chemia and improve wound healing. We hypothesizedthat application of a biodegradable collagen matrix con-taining pro-angiogenic monocytes would improvevascularization of the skin, lead to an increase in skinviability, and reduce necrosis. A trend toward improved

Fig. 3. Vascularity of skin flap. (a)Neovascularization: new capillarygrowth within loose myxoid stromatypical of angiogenesis (>50% vas-cular density); (b) absence of angio-genesis illustrated by lack of newvessels within a myxoid stroma(<10% vascular density). [Color fig-ure can be viewed in the onlineissue, which is available atwileyonlinelibrary.com.]

Fig. 4. Distribution of vascular density among groups. The dorsalportion of each skin flap was divided into four equal-sized seg-ments for histological analysis, resulting in a total of 20 skin flapsexamined in each group of five animals. Green indicates numberof tissue sections with greater than 50% vascular density; yellowindicates number of tissue sections with 20%–50% vascular den-sity; red indicates number of tissue sections with 10%–20% vas-cular density; and blue indicates number of tissue sections withless than 10% vascular density. [Color figure can be viewed in theonline issue, which is available at wileyonlinelibrary.com.]


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vascularity was seen in the treatment group comparedto the two control groups. However, this improved vascu-larity was not associated with improved flap survival,and no differences in skin viability or degree of necrosiswere observed among the groups after 7 days of woundhealing.

Our results suggest that pro-angiogenic monocytesmay result in improved vascularization of the skin flap,or at least of the skip flap environment, but that thisneovascularization was not sufficient to improve woundhealing. Both the quantity and the quality of the mono-cyte therapy may explain this phenomenon. Homologousmonocytes from a single rat donor—rather than autolo-gous monocytes—were embedded in the matrix appliedto all rats in the monocyte group. Autologous monocytetherapy may have been more effective in improving flapsurvival, but due to the animal size, was not a viabletest technique. Homologous monocytes also introducethe risk of immune destruction of the tissue, which inturn could further hinder the wound-healing process. Inclinical practice, it would be desirable to use autologousmonoctye therapy.

In addition, due to animal size, a relatively limitednumber of monocytes—approximately 200–400,000 peranimal—were obtained and embedded in the collagenmatrix. A greater number of autologous monocytes mayhave further acted to create enough new blood vesselgrowth to enable increased skin flap survival.

The experimental methods used to assess the mono-cyte therapy had several potential limitations.Additional changes in duration of healing and route oftreatment administration may have further enhancedthe results. The duration of wound healing observa-tion—7 days following the operation—may have been tooshort to see gross differences in wound healing. A longerduration for wound healing may have further increasedneovascularization and yielded an observable improve-ment in skin viability.

The method of application may have been insuffi-cient to deliver the monocyte therapy effectively.Treatment was limited to the underlying distal portionof the skin flap. Application of i-MonogridTM under theentire flap area may be required in order to allow betterblood flow toward the distal end of the flap. Topicalapplication may be a self-limiting technique for thera-peutic angiogenesis as it forms a physical barrierbetween the flap and the underlining tissue. Injection tothe flap may be a better mode of delivery in order toachieve angiogenesis within the flap. Additional trialswill permit elucidation of the best application technique.

In addition, the complexity of a composite ischemicskin flap model may have been too challenging to betreated with topical monocyte therapy alone. A moresimple ischemic skin flap model—of smaller size or a dif-ferent location—may have been a better approach to testthe efficacy of the treatment. It is also possible that con-tamination hindered the wound healing process. The

surgical operation was completed in a clean, but notsterile, environment, and infection may have adverselyaffected the monocyte therapy. Nonetheless, it is provoc-ative to observe such a degree of increased woundvascularity in the treatment group. The activity of thei-MonogridTM appears promising for improvement of thewound-healing environment.

Finally, it is important to address the potential on-cogenic effect of increasing angiogenesis, especially inpatients suffering from postradiation therapy recur-rences. The potential for adverse outcomes followingcancer treatment by increasing angiogenesis at a resec-tion site remains unknown. However, general practiceremains to maximize the ability for complex wounds toheal. Therefore, monocyte therapy may prove to be auseful adjunct, even in patients with a history of localmalignancy.

CONCLUSIONSDelivery of activated pro-angiogenic monocytes to

an ischemic skin flap tended to improve histologic evi-dence of vascularity without corresponding microscopicor gross evidence of improved flap survival. Theseresults are encouraging regarding the use of monocytesas a potential method of improving vascularization of is-chemic tissue. A greater number of monocytes in eachdose, or injection rather than topical application, mayyield improved results in other applications.

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1. Martinez FO, Gordon S, Locati M, Mantovani A. Transcrip-tional profiling of the human monocyte-to-macrophagedifferentiation and polarization: new molecules and pat-terns of gene expression. J Immunol 2006;177:7303–7311.

2. Mantovani A, Sica A, Locati M. New vistas on macrophagedifferentiation and activation. Eur J Immunol 2007;37:14–16.

3. Crowther M, Brown NJ, Bishop ET, Lewis CE. Microenviron-mental influence on macrophage regulation of angiogene-sis in wounds and malignant tumors. J Leukoc Biol 2001;70:478–490.

4. Mantovani A, Sica A, Locati M. Macrophage polarizationcomes of age. Immunity 2005;23:344–346.

5. Cha J, Falanga V. Stem cells in cutaneous wound healing.Clin Dermatol 2007;25:73–78.

6. Velazquez OC. Angiogenesis and vasculogenesis: inducingthe growth of new blood vessels and wound healing bystimulation of bone marrow-derived progenitor cell mobi-lization and homing. J Vasc Surg 2007;45(Suppl A):A39–A47.

7. Chan RK, Garfein E, Gigante PR, Liu P, Agha RA, MulliganR, Orgill DP. Side population hematopoietic stem cellspromote wound healing in diabetic mice. Plast ReconstrSurg 2007;120:407–411; discussion 412–403.

8. McFarlin K, Gao X, Liu YB, et al. Bone marrow-derived mes-enchymal stromal cells accelerate wound healing in therat. Wound Repair Regen 2006;14:471–478.

9. McFarlane RM, DeYoung G, Henry RA. The design of a pedi-cle flap in the rat to study necrosis and its prevention.Plast Reconstr Surg 1965;35:177–182.


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enhancement of ischemic wound healing by inducement of local angiogenesis

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