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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.


Laryngoscope 120: September 2010                                                    Milch et al.: Angiogenesis in Ischemic Skin Wound
                                                                                                                                        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¡ªblinded to
      Animals (N ? 15) were anesthetized by intraperitoneal         animal group¡ªqualitatively 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.

Laryngoscope 120: September 2010                                             Milch et al.: Angiogenesis in Ischemic Skin Wound
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¡ª
<10%, 10% to 20%, 20% to 50%, >50%¡ªfor 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¡ªdefined as
greater than 50% vascular density¡ªwas 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.]


Laryngoscope 120: September 2010                                        Milch et al.: Angiogenesis in Ischemic Skin Wound
                                                                                                                             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¡ªrather than autolo-          tion site remains unknown. However, general practice
gous monocytes¡ªwere 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¡ªapproximately 200¨C400,000 per              or gross evidence of improved flap survival. These
animal¡ªwere 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¡ª7 days following the operation¡ªmay 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¡ªof smaller size or a dif-            enchymal stromal cells accelerate wound healing in the
                                                                     rat. Wound Repair Regen 2006;14:471¨C478.
ferent location¡ªmay 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.




Laryngoscope 120: September 2010                                        Milch et al.: Angiogenesis in Ischemic Skin Wound
1748

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Enhancement of ischemic wound healing by inducement of local angiogenesis

  • 1. 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
  • 2. 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. Laryngoscope 120: September 2010 Milch et al.: Angiogenesis in Ischemic Skin Wound 1745
  • 3. 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¡ªblinded to Animals (N ? 15) were anesthetized by intraperitoneal animal group¡ªqualitatively 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. Laryngoscope 120: September 2010 Milch et al.: Angiogenesis in Ischemic Skin Wound 1746
  • 4. 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¡ª <10%, 10% to 20%, 20% to 50%, >50%¡ªfor 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¡ªdefined as greater than 50% vascular density¡ªwas 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.] Laryngoscope 120: September 2010 Milch et al.: Angiogenesis in Ischemic Skin Wound 1747
  • 5. 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¡ªrather than autolo- tion site remains unknown. However, general practice gous monocytes¡ªwere 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¡ªapproximately 200¨C400,000 per or gross evidence of improved flap survival. These animal¡ªwere 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¡ª7 days following the operation¡ªmay 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¡ªof smaller size or a dif- enchymal stromal cells accelerate wound healing in the rat. Wound Repair Regen 2006;14:471¨C478. ferent location¡ªmay 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. Laryngoscope 120: September 2010 Milch et al.: Angiogenesis in Ischemic Skin Wound 1748