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The Laryngoscope
V 2010 The American Laryngological,
C
Rhinological and Otological Society, Inc.

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 Key Words: Skin flap necrosis, monocytes,
toward an angiogenic phenotype can be used to angiogenesis.
improve ischemic tissue healing in a rat skin flap Level of Evidence: 5
model. Laryngoscope, 120:1744�C1748, 2010
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. INTRODUCTION
The animals were divided into three groups: the The objective of this study was to evaluate the effi-
monocyte group (N ? 5) received subcutaneous topical cacy of angiogenic monocytes embedded in a
application of 0.1�C0.2 cc of i-MonogridTM, a collagen biodegradable matrix in the treatment of ischemic
gel containing M2 angiogenic monocytes; control wounds. Monocytes have the potential to differentiate
group 1 (N ? 5) received application of cell-free colla- into different functional phenotypes based on their
gen; and control group 2 (N ? 5) received no treat- microenvironment.1,2 The M2 alternative pathway of
ment. Skin flaps were stapled in place and observed monocyte differentiation refers to the pro-angiogenic
for wound ischemia and necrosis of the skin flap. One
phenotype of monocytes.3,4 This study examined the
week postoperatively, skin and underlying muscle
were harvested for histologic analyses. potential of monocytes directed toward their angiogenic
Results: No macroscopic differences in wound (M2) phenotype to enhance ischemic wound healing by
healing or microscopic differences in skin viability increasing angiogenesis.
were observed. However, the monocyte group showed Local delivery of progenitor cells to ischemic
significantly greater vascular improvement than C1 wounds has been demonstrated to enhance wound heal-
(P ? .047, v ? 3.96), and a trend toward greater vas- ing.5�C7 For example, McFarlin et al.8 showed that
cular improvement than C2 (P ? .103, v ? 2.67). systemic administration of bone marrow-derived mesen-
Conclusions: Delivery of activated pro-angio- chymal stromal cells improved wound healing in rats.
genic monocytes to an ischemic skin flap tended to However, the availability of stem cells for therapeutic
improve histologic evidence of vascularity without
use is limited, and the use of pro-angiogenic monocytes
corresponding microscopic or gross evidence of
improved flap survival. These results are encouraging could prove to be a viable alternative. Monocytes are
regarding the use of monocytes as a potential method readily available by separation from peripheral blood
of improving vascularization of ischemic tissue. and can be easily obtained in large numbers from
patients. Monocytes may provide a source for therapeu-
tic angiogenic cells, and have the ability improve skin
flap surgery outcomes and the treatment of ischemic
From the Boston University School of Medicine (H.S.M.), Boston, wounds. The therapeutic applications are many, as
Massachusetts, U.S.A.; Moma Therapeutics ( S . Y. S .), Brighton, increased angiogenesis can ideally make healing more
Massachusetts, U.S.A.; Department of Pathology and Laboratory
Medicine (S.H.), Boston Medical Center, Boston, Massachusetts, U.S.A.; rapid and robust, particularly in environments with
Department of Otolaryngology�CHead and Neck Surgery (J.H.S.), Boston potentially compromised healing. These would include in
University School of Medicine, Boston, Massachusetts, U.S.A. people who smoke, those with prior radiation or other
Editor��s Note: This Manuscript was accepted for publication April
26, 2010.
harmful treatments, individuals with comorbidities such
Financial disclosure information: Shai Y. Schubert, PhD, is Presi- as diabetes mellitus, and those in whom skin flap design
dent of Moma Therapeutics, Brighton, MA; Jeffrey H. Spiegel, MD, is on was suboptimal.
the Advisory Board of Moma Therapeutics, Brighton, MA. Evaluation of the potential of angiogenic monocytes
The authors declare no conflicts of interest.
This was a TRIO Section Meeting Oral Presentation. embedded in 3D matrices for the treatment of ischemic
Send correspondence to Dr. Jeffrey H. Spiegel, Boston University wound healing was carried out with two specific goals: 1)
School of Medicine, 830 Harrison Avenue, Suite 1400, Boston, MA 02118. to determine the ability of collagen gel embedded with
E-mail: Jeffrey.Spiegel@bmc.org
M2 angiogenic monocytes to reduce ischemic injury (ne-
DOI: 10.1002/lary.21068 crosis) in single-pedicle dorsal skin flap injury in rats;

Laryngoscope 120: September 2010 Milch et al.: Angiogenesis in Ischemic Skin Wound
1744

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 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 matrix of collagen and polycaprolactone particles (PCL)
with M2 angiogenic monocytes on angiogenesis. 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
METHODS was hypothesized that autologous monocytes directed toward
In this study, monocytes polarized toward the M2 angiogenic their angiogenic (M2) phenotype would enhance ischemic
phenotype were embedded in i-MonogridTM, a biodegradable wound healing by increasing angiogenesis.

1745

Fig. 2. Viability of skin klap. (a) Full
thickness epidermis with underlying
dermis showing functional adnexal
structures (>50% viability); (b) ne-
crotic 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 Institu- ation of wound healing was monitored using analysis of images
tional IACUC committee at Boston University School of of the injury taken every 24 hours during the course of the
Medicine. i-MonogridTM was provided by Moma Therapeutics. study.
Monocytes were separated from rat blood obtained by homolo- At day 7 postoperation, the skin flaps were harvested for
gous collection. Following gradient centrifugation of blood [50% histologic analyses. For analysis, the flap was divided into prox-
in phosphate-buffered saline (PBS)] on Histopaque-1077, mono- imal and distal portions. The proximal portion was the area of
cytes were separated from the mononuclear cell fraction the flap closer to the scapula and was expected to behave simi-
employing a negative isolation method based on the depletion of lar to normal tissue. In contrast, severe ischemia and necrosis
nonmonocytes by their binding to antibody coated magnetic were expected in the distal portion of the flap. To evaluate
beads (Miltenyi Biotec, Auburn, CA). wound angiogenesis, vascularity in the distal sections of the
The separation results in high purity (>90%) of untouched skin flap was measured at day 7 postoperation. Wound healing
monocytes. A total of 5 cc of blood were taken from a donor rat was characterized by evaluation of skin flap recovery using
approximately 250 g in size. Monocytes were incubated in 100 both visual parameters and histology of the wound at day 7
nM adenosine in PBS, 2.5 mM EDTA for 1 hour prior to addi- postoperation.
tion of collagen. To evaluate wound angiogenesis, a pathologist��blinded to
Animals (N ? 15) were anesthetized by intraperitoneal animal group��qualitatively assessed the density of neocapilla-
injection of xylazine and ketamine (40�C80 mg/kg ketamine and rization within each skin flap. Four representative tissue
5�C10 mg/kg xylazine), with perioperative analgesia with subcu- sections were evaluated from each animal, such that a total of
taneous buprenorphine at the time of surgery and doses of 0.05 20 tissue sections were assessed in each group of five animals.
mg/kg subcutaneously every 12 hours for 2 days postoperatively. Vascularization was measured on a scale of 0 to 100, where the
A caudally based 9 ? 3 cm dorsal skin/panniculus carnosus flap least vascularized graft was defined as 0, and the most vascu-
was raised with the two constant sacral axial vessels systemati- larized graft was defined as 100. Grafts were given a score
cally cut. This skin flap design is a modification of the single based on the level of vascularization within this scale. Improved
pedicle dorsal skin flap previously published as a model for is- vascularity was defined as greater than 50% vascular density of
chemic skin flap healing.9 The caudal border of the flap was the skin flap relative to the degree of vascularity in all tissue
marked at 1 cm below the posterior iliac crests. samples. Chi-squared analyses were used to determine differen-
The animals were divided into three groups: a monocyte ces in vascularity among groups.
treated group and two control groups. In the distal end of the
skin flap, the monocyte group (N ? 5) received subcutaneous
topical application of 0.1�C0.2 cc of i-MonogridTM, a collagen gel
RESULTS
containing M2 angiogenic monocytes. Approximately 200�C
400,000 monocytes were delivered to each animal in the treat- Macroscopic
ment group. Control group 1 (C1, N ? 5) received subcutaneous Necrosis was observed in all animals by 3�C5 days
topical application of cell free collagen. Control group 2 (C2, N following the operation, marked by hardening of the
? 5) received no applications. All collagen and monocyte appli-
skin, darkening, and loss of hair. At day 7, necrotic tis-
cations occurred while the animals were sedated, shortly after
sue encompassed one-quarter to one-half of the distal
operation and before wound closure. The use of type 1 human
collagen as the carrier for the monocytes was used to minimize portion of all skin flaps (Fig. 1). The extent of necrosis
the risk of adverse reactions. The skin flaps of all animals were among individual animals was not diminished within
then repositioned and stapled in place. any one particular group. These results suggest that is-
After the surgical procedure, the animals were returned to chemia led to poor wound healing in all rats, regardless
individual cages and received food and water ad libitum. Evalu- of treatment with pro-angiogenic monocytes.

1746

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

Histology significant difference in vascular improvement between
Histologic evaluation of the skin flap focused on two the two control groups (P ? .705, v ? 0.143).
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 underlying DISCUSSION
dermis, and intact adnexal structures (Fig. 2a). Nonvi- The goal of the monocyte treatment was to increase
able skin showed evidence of a necrotic epidermis and neovascularization of the skin flap in order to reduce is-
dermis, and no functional adnexal structures (Fig. 2b). chemia and improve wound healing. We hypothesized
Vascularity of the skin was evaluated based on the that application of a biodegradable collagen matrix con-
degree of neocapillarization of the skin and subcutis taining pro-angiogenic monocytes would improve
(Fig. 3). Tissue vascularity did not appear to depend on vascularization of the skin, lead to an increase in skin
skin viability, that is, a skin section with minimal intact viability, and reduce necrosis. A trend toward improved
dermis and epidermis often corresponded with an abun-
dance of underying granulation tissue and new blood
vessel growth. Conversely, an intact dermis and epider-
mis 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).

Viability
No group differences were observed in skin flap
viability.

Vascularity
A trend toward improved vascularity��defined as
greater than 50% vascular density��was observed in the
monocyte group. The monocyte group exhibited more
than double the amount of vascular improvement as the
Fig. 4. Distribution of vascular density among groups. The dorsal
two control groups (Fig. 4). The monocyte group showed
portion of each skin flap was divided into four equal-sized seg-
significantly greater vascular improvement than C1 ments for histological analysis, resulting in a total of 20 skin flaps
(P ? .047, v ? 3.96), and a trend toward greater vascular examined in each group of five animals. Green indicates number
improvement than C2, although this comparison was not of tissue sections with greater than 50% vascular density; yellow
statistically significant (P ? .103, v ? 2.67). A three-way indicates number of tissue sections with 20%�C50% vascular den-
sity; red indicates number of tissue sections with 10%�C20% vas-
comparison also showed a trend toward differences in cular density; and blue indicates number of tissue sections with
vascular improvement (P ? .092), although this differ- less than 10% vascular density. [Color figure can be viewed in the
ence was not statistically significant. There was no online issue, which is available at wileyonlinelibrary.com.]

1747

vascularity was seen in the treatment group compared surgical operation was completed in a clean, but not
to the two control groups. However, this improved vascu- sterile, environment, and infection may have adversely
larity was not associated with improved flap survival, affected the monocyte therapy. Nonetheless, it is provoc-
and no differences in skin viability or degree of necrosis ative to observe such a degree of increased wound
were observed among the groups after 7 days of wound vascularity in the treatment group. The activity of the
healing. i-MonogridTM appears promising for improvement of the
Our results suggest that pro-angiogenic monocytes wound-healing environment.
may result in improved vascularization of the skin flap, Finally, it is important to address the potential on-
or at least of the skip flap environment, but that this cogenic effect of increasing angiogenesis, especially in
neovascularization was not sufficient to improve wound patients suffering from postradiation therapy recur-
healing. Both the quantity and the quality of the mono- rences. The potential for adverse outcomes following
cyte therapy may explain this phenomenon. Homologous cancer treatment by increasing angiogenesis at a resec-
monocytes from a single rat donor��rather than autolo- tion site remains unknown. However, general practice
gous monocytes��were embedded in the matrix applied remains to maximize the ability for complex wounds to
to all rats in the monocyte group. Autologous monocyte heal. Therefore, monocyte therapy may prove to be a
therapy may have been more effective in improving flap useful adjunct, even in patients with a history of local
survival, but due to the animal size, was not a viable malignancy.
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 CONCLUSIONS
clinical practice, it would be desirable to use autologous Delivery of activated pro-angiogenic monocytes to
monoctye therapy. an ischemic skin flap tended to improve histologic evi-
In addition, due to animal size, a relatively limited dence of vascularity without corresponding microscopic
number of monocytes��approximately 200�C400,000 per or gross evidence of improved flap survival. These
animal��were obtained and embedded in the collagen results are encouraging regarding the use of monocytes
matrix. A greater number of autologous monocytes may as a potential method of improving vascularization of is-
have further acted to create enough new blood vessel chemic tissue. A greater number of monocytes in each
growth to enable increased skin flap survival. dose, or injection rather than topical application, may
The experimental methods used to assess the mono- yield improved results in other applications.
cyte therapy had several potential limitations.
Additional changes in duration of healing and route of
treatment administration may have further enhanced BIBLIOGRAPHY
the results. The duration of wound healing observa- 1. Martinez FO, Gordon S, Locati M, Mantovani A. Transcrip-
tion��7 days following the operation��may have been too tional profiling of the human monocyte-to-macrophage
short to see gross differences in wound healing. A longer differentiation and polarization: new molecules and pat-
duration for wound healing may have further increased terns of gene expression. J Immunol 2006;177:7303�C7311.
2. Mantovani A, Sica A, Locati M. New vistas on macrophage
neovascularization and yielded an observable improve- differentiation and activation. Eur J Immunol 2007;37:
ment in skin viability. 14�C16.
The method of application may have been insuffi- 3. Crowther M, Brown NJ, Bishop ET, Lewis CE. Microenviron-
cient to deliver the monocyte therapy effectively. mental influence on macrophage regulation of angiogene-
Treatment was limited to the underlying distal portion sis in wounds and malignant tumors. J Leukoc Biol 2001;
70:478�C490.
of the skin flap. Application of i-MonogridTM under the 4. Mantovani A, Sica A, Locati M. Macrophage polarization
entire flap area may be required in order to allow better comes of age. Immunity 2005;23:344�C346.
blood flow toward the distal end of the flap. Topical 5. Cha J, Falanga V. Stem cells in cutaneous wound healing.
application may be a self-limiting technique for thera- Clin Dermatol 2007;25:73�C78.
6. Velazquez OC. Angiogenesis and vasculogenesis: inducing
peutic angiogenesis as it forms a physical barrier the growth of new blood vessels and wound healing by
between the flap and the underlining tissue. Injection to stimulation of bone marrow-derived progenitor cell mobi-
the flap may be a better mode of delivery in order to lization and homing. J Vasc Surg 2007;45(Suppl A):
achieve angiogenesis within the flap. Additional trials A39�CA47.
7. Chan RK, Garfein E, Gigante PR, Liu P, Agha RA, Mulligan
will permit elucidation of the best application technique.
R, Orgill DP. Side population hematopoietic stem cells
In addition, the complexity of a composite ischemic promote wound healing in diabetic mice. Plast Reconstr
skin flap model may have been too challenging to be Surg 2007;120:407�C411; discussion 412�C403.
treated with topical monocyte therapy alone. A more 8. McFarlin K, Gao X, Liu YB, et al. Bone marrow-derived mes-
simple ischemic skin flap model��of smaller size or a dif- enchymal stromal cells accelerate wound healing in the
rat. Wound Repair Regen 2006;14:471�C478.
ferent location��may have been a better approach to test 9. McFarlane RM, DeYoung G, Henry RA. The design of a pedi-
the efficacy of the treatment. It is also possible that con- cle flap in the rat to study necrosis and its prevention.
tamination hindered the wound healing process. The Plast Reconstr Surg 1965;35:177�C182.

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