BONE MARROW MONONUCLEAR CELLS STIMULATEANGIOGENESIS WHEN TRANSPLANTED INTO SURGICALLYINDUCED FIBROCOLLAGENOUS TUNNELS: RESULTS FROMA CANINE ISCHEMIC HINDLIMB MODEL

LUIS PADILLA, M.D.,1,2,3* EDGAR KROTZSCH, Ph.D.,4 ANABEL S. DE LA GARZA, M.D.,1 SIEGFRIED FIGUEROA, M.D.,1

JUAN RODRIGUEZ-TREJO, M.D.,5 GERARDO AVILA, M.D.,1 PAUL SCHALCH, M.D.,1 IGNACIO ESCOTTO, M.D.,5

GERMAN GLENNIE, D.V.M.,6 FERNANDO VILLEGAS, M.D.,3 and MAURICIO DI SILVIO, M.D., F.A.C.S.1

Progenitor cell transplantation has been considered as a potential angiogenesis therapy for the ischemic hindlimb. In this work we per-formed an ischemic hindlimb model in dogs. We ligated the middle sacra and the external right iliac arteries. After 7 days, the femoral ar-tery was ligated and removed, and three Silastic1 tubes were inserted into the gracilis muscle to create fibrocollagenous tunnels. AfterSilastic1 implantation, we administered saline or granulocyte colony stimulating factor (G-CSF) subcutaneously daily during 5 days. Four-teen days after device positioning we transplanted bone marrow mononuclear cells (BMMC) into the tunnels previously formed by Silastic1

tube reaction. Twenty-eight days later, contrasted angiographies were performed and angiographic scores were calculated. Also, vesselsand endothelial cells and proliferating cells were identified by immunochemistry of muscle sections. Results demonstrated that BMMCtransplantation enriched by G-CSF administration significantly stimulates angiogenesis in the ischemic hindlimb, and more than BMMCtransplantation alone. Transplantation of progenitor cells in an appropriate extracellular matrix is a potential therapy for hindlimbischemia. VVC 2006 Wiley-Liss, Inc. Microsurgery 27:91–97, 2007.

Patients with peripheral occlusive arterial disease

(POAD) are candidates for distal revascularization when

they present: a) incapacitating claudication that fails to

respond to conservative measures and generally has better

postoperative results because the arterial disease is less

severe or b) critical ischemic disease (ankle-brachial index

<0.30 or <40 mmHg systolic pressure at the ankle, ische-

mic pain at rest, or distal ischemic atrophic lesions).1 Dor-

mandy et al.,2 reported that distal revascularization in

patients with critical ischemic disease reduced the percent-

age of major amputations by 11–4%, however, not all

patients are candidates for this procedure because severe

ischemia presents with multiple distal stenotic lesions, thus

making distal revascularization attempts unsuccessful. If

other comorbidities such as diabetes mellitus, coronary ar-

tery, or carotid disease are present, the possibility of sav-

ing the extremity is even further reduced, because of the

unacceptable risk of intra- and postoperative complica-

tions.3 This group of patients has therefore a major ampu-

tation likelihood of 10–40%.4 The concept of therapeutic

angiogenesis emerges as a result of the search for alterna-

tive treatments for ischemic diseases such as POAD.5–9

This new term encompasses a therapeutic strategy aimed

at promoting the development of small capillaries, thus

improving collateral blood flow in ischemic tissues.

Angiogenesis and arteriogenesis are complex proc-

esses involving a sequence of steps, which include a)

migration of endothelial cells of the existing vessels

through breakdown of the extracellular matrix, b) endo-

thelial cell proliferation, and c) anastomosis with other

newly formed vessels, resulting in functional capillary

networks.10,11 This process can be stimulated by the

administration of angiogenic growth factors, such as basic

fibroblast growth factor (bFGF)12 and vascular endothelial

growth factor (VEGF),13 as well as more ‘‘upstream’’

transcription factors such as early growth response-1

(Egr-1).14 In addition, gene transfection for protein codifi-

cation can also be used as it plays an important role in

endothelial progenitor cells (EPCs) maturation process

and the maintenance of the vascular system.15 In contrast

to angiogenesis and arteriogenesis, postnatal vasculogene-

sis is the development of new capillary networks from

EPCs, which are present within the reserve of the bone

marrow mononuclear cells (BMMC),10 as well as in the

peripheral blood in adults.11 EPCs migrate to and incor-

porate into neovascular sites to complete their differentia-

tion into endothelial cells, thus giving rise to new capil-

lary networks.16,17 The natural ability of BMMC to produce

precursors that give rise to diverse cellular lineages, includ-

1Department of Microsurgery, Centro Medico Nacional ‘‘20 de Noviembre’’,I.S.S.S.T.E., Mexico City, Mexico2Department of Experimental Surgery, Centro Medico Nacional ‘‘20 deNoviembre’’, I.S.S.S.T.E., Mexico City, Mexico3Microsurgery Unit, Department of Surgery, School of Medicine, U.N.A.M.,Mexico City, Mexico4Connective Tissue Laboratory, Biomedical Research Division, Centro Med-ico Nacional ‘‘20 de Noviembre’’, I.S.S.S.T.E., Mexico City, Mexico5Department of Angiology and Vascular Surgery, Centro Medico Nacional‘‘20 de Noviembre’’, I.S.S.S.T.E., Mexico City, Mexico6Division of Laboratory Animal Resources, Centro Medico Nacional ‘‘20 deNoviembre’’, I.S.S.S.T.E., Mexico City, Mexico

Grant sponsor: Sociedad Mexicana de Investigacion Biomedica A.C.*Correspondence to: Luis Padilla, M.D., Millet no. 83-205, Col. ExtremaduraInsurgentes, Mexico City, 03740, Mexico.E-mail: lpadilla@issste.gob.mx

Received 3 October 2005; Accepted 27 August 2006

Published online 21 December 2006 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/micr.20289

VVC 2006 Wiley-Liss, Inc.

ing EPCs, which in turn release several angiogenic factors

and cytokines that stimulate the formation of new vessels,

provides the framework for a potential new strategy for

the induction of therapeutic angiogenesis.18,19 In recent

preclinical and clinical studies where autologous implanta-

tion of BMMC was used, a significant increase in the for-

mation of collateral vessels was demonstrated.20 Recent

studies suggest that BMMC CD34� stimulate CD34þ

cells, which synthesize VEGF, bFGF, and angiopoyetin-

1.17 These factors are known to participate in the process

of angiogenesis, although their exact mechanism of action

is not yet fully understood. In vitro studies using BMMC

CD34þ under hypoxic conditions have clearly demon-

strated the capacity of these cells to release angiogenic

factors and induce the formation of new vessels.21 Several

studies involving animal models show that the implanta-

tion of autologous of BMMC in the cornea,22 ischemic

limbs, and myocardium in rats23,24 and pigs,20 promotes

the formation of collateral vessels with improvement of

the regional blood flow. Additionally, the incorporation of

EPCs has been detected in capillaries formed de novo and

the local concentrations of angiogenic factors and cyto-

kines such as interleukin 1-beta (IL-lb) and tumor necrosis

factor alpha (TNFa) were increased.20 Recombinant

human granulocyte colony stimulating factor (rhG-CSF)

has also been shown to contribute to the formation of new

capillary networks. Reports suggest that it has a stimulat-

ing effect on the proliferation and migration of endothelial

cells, thereby enhancing collateral vessel formation. RhG-

CSF was initially described as a factor responsible for con-

trolling the proliferation and maturation of granulocytes

and their precursors, in addition to regulating neutrophil

activity.25,26 Previous studies also indicate that rhG-CSF

increases the number of EPCs in both the BM and in pe-

ripheral blood.27

Studies in vitro and in vivo have demonstrated that he-

matopoietic and endothelial cells are derived from a com-

mon cell, which expresses the surface antigen CD34, as

well as tyrosine kinase-like receptors (Tie, Tek, and Flk-1).

Shi et al. have confirmed that BM-derived CD34 cells can

colonize the endothelial surfaces of vascular prosthesis and

can differentiate into actual endothelial cells.28

Using a model of limb ischemia in rats, our group

demonstrated that angiogenesis can be induced more

effectively through the transplantation of BMMC in fibro-

collagenous tunnels, which provide a sort of ‘‘scaffold’’

for the differentiation and survival of BMMC, and facili-

tate the interactions of growth factors, cytokines, and

cells involved in the process of ischemia, reperfusion,

and healing.29 Our current study utilizes a model of suba-

cute ischemia in dog hindlimbs. We evaluated the forma-

tion of new vessel networks after transplanting autolo-

gous BMMC in previously induced fibrocollagenous tun-

nels in conjunction with rhG-CSF administration.

MATERIALS AND METHODS

Twenty mongrel dogs weighing 18–22 kg were fed

ad libitum with standard dog chow (Purina Mainstay,

Ralston Purina, Mexico) and treated in accordance with

institutional animal care guidelines from the Ethics and

Research Committee of the Centro Medico Nacional ‘‘20

de Noviembre’’, Mexico City, the General Health Legis-

lation on Laboratory Animals Research and the guide for

use of laboratory animals of the National Institutes of

Health, Bethesda, MD, USA.

Surgical Technique

After inducing general anesthesia (xylacine�HCl1 mg/kg IM, Zoletil 50, Bayer, Mexico City and tiletha-

mine�HCl 125 mg/zolazepam�HCl 125 mg 7 mg/kg IV,

Virbac, Carros, France) the bladder was emptied with a

catheter. Each dog was placed in reverse Trendelenburg

position and a 10 cm trocar was introduced through a 2

cm periumbilical incision, through which penumoperito-

neum was established and a laparoscope was advanced

(Contec Medical, Israel). An additional two trocars (10

and 5 cm) were then placed 4 cm below and lateral from

the first (right and left), and a 4th trocar was then intro-

duced left of the first, 6 cm below, under direct visualiza-

tion. The abdominal aorta trifurcation was then dissected

and the median sacral artery was ligated using 3-0 silk

suture (Atramat, Mexico). The median sacral artery was

then excised by clipping the proximal and distal ends

(Ethicon Endo-surgery clips, Johnson and Johnson, NJ,

USA). The external iliac artery was then stapled across

its proximal and distal ends, and was excised. Trocars

were removed and incisions sutured with polypropylene

3-0 (Ethicon, Johnson and Johnson, NJ, USA). During

the following 3 days, penicillin G (800,000 U Q12 h,

Ivax Pharmaceuticals, Mexico) and metamizol (50 mg Q12

h, Pisa, Mexico) were administered for postsurgical prophy-

laxis and pain control.

Seven days later, laparoscopy was again performed; a

segment of the femoral artery was dissected free, and

after performing proximal and distal ligation, excised.

During this second procedure, an incision over the inner

thigh was made and Silastic1 tubes (Dow Corning, Mid-

land, MI, USA) with an outside diameter of 0.8 mm, and

22, 24, and 26 cm in length respectively, were inserted

through the inner thigh muscles with the aim of inducing

the formation of ‘‘u’’ shaped fibrocollagenous tunnels.

Polyester mesh (3.5 cm in length) was attached to the dis-

tal ends of each tube so as to facilitate localization later

on and they were fixed to the surrounding muscle using

nylon 5-0 (Le Mare Internacional, Mexico) to avoid dis-

placement. (Fig. 1). The incision was closed using catgut

3-0 (Atramat, Mexico).

92 Padilla et al.

Microsurgery DOI 10.1002/micr

During the following 5 days, saline solution (SS) or

granulocyte colony stimulating factor (5 lg/kg/day rhG-

CSF, Filgrastim, Roche, Mexico) were administered sub-

cutaneously in the scapular region 3to the designated

group (10 animals each).

Fourteen days after the insertion of the Silastic1 tubes,

a small incision in the skin of the inner right leg was

made and all tubes were removed after cutting below the

polyester mesh and gentle traction. Before cell transplanta-

tion, each tunnel was washed with heparin diluted in saline

(1:20,000). Finally, the tunnel ends were sealed at the

polyester mesh level and 2.5 3 107 BMMC or SS (con-

trol) were administered only once (10 animals each, 5

dogs for BMMC, and 5 for G-CSF/BMMC groups). The

distal ends were sealed and the incision was closed using

nylon 3-0. Prophylactic antibiotics and analgesics were

administered after each procedure for 3 consecutive days.

BM Mononuclear Cell Isolation

Dogs receiving BM transplant underwent surgical ex-

posure of the left humerus and with the aid of a micro-

drill, a 2-mm perforation was made and BM was aspirated

using a heparinized syringe attached to a catheter. Thirty

milliliters of BM aspirate were obtained from each animal.

The bone defect was sealed with surgical wax and the

incision was closed using 3-0 polyglycolic acid suture.

BMMC were isolated with Ficoll gradient (Histopaque

1.077, Sigma, St. Louis, MO, USA) and cell viability was

determined by means of the trypan blue exclusion dye tech-

nique. A total dose of 2.53 107 cells with over 95% viability

were resuspended in 1.8 ml of RPMI-1640 (HyClone, Logan,

UT, USA) and injected into the fibrocollagenous tunnels, as

described; proportional distribution of the cells was calcu-

lated according to the length of each tunnel.

Angiographic Studies

Forty-nine days after the initial procedure (28 days

after cell transplantation), contrast angiography was per-

formed by cannulating the abdominal aorta above the

bifurcation of the iliac arteries. The arteries were main-

tained with 10 ml of a 1:1 heparin/lidocaine solution im-

mediately before than 9 ml of contrast medium (Optiray-

30, Mallinckrodt, Quebec, Canada) were administrated

under fluoroscopy. Then an X-ray image of both hind-

limbs was obtained. A blinded observer placed a trans-

parent acetate over the X-ray image and copied the vas-

cular pattern with ink. The number of intersections was

counted using a centimeter scaled paper in an area of

10 3 10 cm, as previously described (angiographic score).7

Dogs were subsequently sacrificed by asphyxia with CO2.

Histological Analysis

Muscle biopsies containing fibrocollagenous tunnels

were obtained from each animal, embedded in a tissue

freezing medium, and snap frozen in liquid nitrogen.

Samples were then maintained at �708C until processing.

Serial 6-lm-thick cryocuts were done and stained accord-

ing to Herovici technique.30

Endothelial Cell and PCNA Immunostaining

Biopsies from the central portion of the inner thigh

muscles were obtained from both limbs after angiogra-

phy; the contralateral limb serving as control. Tissues

were fixed with neutral formalin for 24 h and then em-

bedded in paraffin. Five micrometer sections were done

and paraffin was removed from the tissue samples. Sec-

tions were then immunostained for Griffonia simplicifoliaisotype B4 lectin (GSL-isoB4) and proliferating cell nu-

clear antigen (PCNA) to detect microvasculature31 and

proliferating cells.32 The endogenous peroxidase was

inhibited with 9:1 hydrogen peroxide/methanol for 15

min, and sections were then treated by heat-induced epi-

tope retrieval technique with citrate buffer solution,

according to the manufacturer’s instructions (Zymed, San

Francisco, CA, USA). Immunohistochemical analysis of

the 1:100 GSL-isoB4, Vector, CA, USA) or Zymed1

Figure 1. Schematic representation of the surgical technique for

induction of ischemia and fibrocollagenous tunnel formation. The

three Silastic1 tubes are inserted through the inner thigh muscles

of the dog.

BMMC Induces Angiogenesis in Fibrocollagenous Tunnels 93

Microsurgery DOI 10.1002/micr

PCNA staining kit (Clone PC-10, Zymed, San Francisco,

CA, USA) was performed. For the lectin assay we used

the biotin-avidin-peroxidase method, with aminoethilcar-

bazole development and counter staining the nuclei with

Mayer’s Hematoxilin, as previously described.29

Capillary/fiber ratios were calculated by counting

capillaries and muscle fibers at 4003 in three random

linearly aligned fields in the central portion of the tissue

in two serial sections. To establish the presence of prolif-

erating cells, the percentage of immunoreactive nuclei

was determined at 10003 by counting 100 nuclei in

randomized fields in the central portion of the section in

two serial sections per specimen. Histology and immuno-

histochemistry were performed in a blinded fashion.

Statistical Analysis

Angiographic scores, capillary/fiber, and the percent-

age of PCNA immunoreactive nuclei ratios were

expressed as means 6 SD by a nonparametric variance

analysis according to the Kruskal–Wallis test and the dif-

ference, group vs. group, was then evaluated by Wil-

coxon test to know any significant difference. Results

were considered statistically significant for P values

�0.05.

RESULTS

Angiographic Analysis

The number of vascular intersections (Fig. 2), as a func-

tion of venous and arterial reconnection, was recorded and

expressed as the mean angiographic score (MAS), (Fig. 3).

MAS for the different groups were: G-CSF/BMMC 2.13 60.31, BMMC 1.76 6 0.38, G-CSF 1.35 6 0.27, SS 1.16 60.66, and the control group 0.8 6 0.42. With respect to the

control group, its MAS was significantly lower when com-

pared to all of the other groups (P � 0.043. Fig. 3). Also,

significant differences were observed among treatments,

particularly when SS and G-CSF groups were compared

with G-CSF/BMMC group (P � 0.05. Fig. 3).

It is important to note that all of the treated groups

showed a significant increase in the angiogenic score

when compared to the contralateral, untreated limbs

belonging to animals in the same group.

Microvascular and Endothelial Cell Proliferation

To identify the degree of angiogenesis in small ves-

sels, we employed immunohistochemical methods for the

detection of endothelial cells and endothelial cell prolifer-

ation: Griffonia simplicifolia isotype B4 lectin and

PCNA, respectively. Capillary/fiber ratios were calculated

and differences among groups were found not to be stat-

Figure 2. Angiographic images and their vascular pattern. An area of 10 3 10 cm in a centimeter scaled paper was used to calculate the

number of intersections, which is expressed as an angiographic score. Panels indicate treatments, saline solution (SS), recombinant

human granulocyte colony stimulating factor (G-CSF), bone marrow (BM), and the combined treatment of G-CSF and BM.

94 Padilla et al.

Microsurgery DOI 10.1002/micr

istically significant (data not shown). PCNA positive en-

dothelial nuclei were analyzed based on their microana-

tomic localization of endothelial cells.28 We observed a

twofold increased proliferation in the group treated with

G-CSF when compared with control group (P < 0.003).

Also, there were significant differences among G-CSF

group vs. SS and BMMC, P values were 0.03 and 0.02,

respectively (Fig. 4).

Ex-Vivo Type I/III Collagen Expression

Fibrocollagenous tunnels are extracellular matrix

structures formed as a result of an inflammatory reaction

unleashed by an immune response to the silicone tubes

previously inserted. These tunnels are important for

BMMC deposition, since it is recognized that mononu-

clear cells display membrane ligands for extracellular

matrix (integrins).33,34

We determined the proportion of type I and type III

collagen in the tunnels, as well as whether endothelial

cells are actually deposited in the lumen. Results showed

that the extracellular matrix in the tunnels consists pre-

dominantly of thick fibers of type I collagen (magenta)

lined by a fine layer of type III collagen fibers (blue) in

the inner region of the structure (Fig. 5). There were no

GSL-isoB4 reactive marks to be found in the lumen of

the tunnels (data not shown).

DISCUSSION

Hindlimb ischemia is characterized by a decrease in

blood flow and subsequent blood vessel (BV) loss. The

objective of therapeutic angiogenesis is to improve blood

distribution in the ischemic region through new vessel

formation and/or vessel growth.5 It has been demon-

strated that BM cells stimulate angiogenesis when they

are locally administered.13,16,18,20 Our group previously

demonstrated that BM cells administered into fibrocollag-

enous tunnels increase angiographic scores in rats.29 In

Figure 3. Angiographic score. Bars represent the mean 6 S.D. for

each treatment group. Control group or healthy limb (Ctrl, n ¼ 5),

saline solution (SS, n ¼ 5), G-CSF group (n ¼ 5), bone marrow

group (BM, n ¼ 5), and G-CSF/BM group (n ¼ 5).

Figure 4. Endothelial cell proliferation. Bars represent the mean 6S.D. for each treatment group. Control group or healthy limb, saline

solution (SS), G-CSF group, bone marrow group (BM), and G-CSF/

BM group.

Figure 5. Ex vivo microphotography showing a fibrocollagenous tun-

nel inside of the muscle (red). The structure consists predominantly

of thick fibers of type I collagen (magenta, Clg I) lined by a fine layer

of type III collagen fibers (blue, Clg III) in the inner region. Blood ves-

sels appear besides the tunnel (BV). Bar represents 500 lm. [Color

figure can be viewed in the online issue, which is available at www.

interscience.wiley.com.]

BMMC Induces Angiogenesis in Fibrocollagenous Tunnels 95

Microsurgery DOI 10.1002/micr

this work we performed a hindlimb ischemic model in

dogs, since phylogenetically canines resemble humans

more than rats do. We established ischemia through par-

tial limb artery resection and we stimulated fibrocollage-

nous tunnel formation by controlled foreign body reaction

through silicone tube insertion. These porous tunnels

were made of extracellular matrix, mainly type I collagen

(Fig. 5) and they were not endothelialized since we could

not observe reactivity against Griffonia simplicifolia iso-

type B4 lectin in the lumen of tunnels (data not shown).

Angiogenesis however, was improved after using G-CSF,

BMMC, or a combination of both. It was also observed

that treatment with G-CSF/BMMC (2.13 6 0.31 vs. 0.80

6 0.42 contralateral limb) obtained the highest angio-

graphic score. Based on the above data it is possible that

G-CSF exerts a synergistic effect on BM cells to improve

angiogenesis. This conclusion is supported by the sys-

temic changes reported after cytokine administration27

where neutrophils are increased when patients are treated

with G-CSF. Nevertheless, BMMC by themselves are a

source of cytokines,20 thus there was not a significant sta-

tistical difference between angiographic score in this

group and the G-CSF/BMMC group (Fig. 3). Further-

more, G-CSF exhibits the ability to mobilize hematopoi-

etic cells from the BM to the peripheral blood through

reduction of expression of some adhesion molecules.34

This information supports conclusions drawn from the

results obtained from the comparison of the G-CSF/

BMMC and BMMC groups, considering that previously

G-CSF-treated BM likely mobilized hematopoietic as

well as EPCs to the peripheral blood.

At day-49 microvasculature of each group did not show

changes, since the capillary/fiber ratio was similar among

them. This result could be due to the fact that when the bi-

opsy was obtained, at day 49, the macrovasculature was al-

ready structured and the ischemia was controlled (Fig. 2)

which is in accordance to previous data.29 However, endo-

thelial cell proliferation changed only when G-CSF group

was compared to controls, SS, or BMMC, perhaps due to

the effects of the growth factor on cells (Fig. 4). We

believe that the timing of the biopsy was responsible, since

the microvasculature, nevertheless G-CSF group.

This work supports the results previously obtained

and also indicates that angiogenesis is present before day

28 and 49 in rats29 and dogs, respectively. More impor-

tantly, these studies have prepared the preclinical frame-

work for a controlled phase I trial in humans.

ACKNOWLEDGMENTS

The authors are grateful to Mathematician Jorge Galicia

for statistical analysis, Brenda Farrington, MD for English

translation, and Juan Carlos Bustamente, MD for biblio-

graphic assistance.

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Microsurgery DOI 10.1002/micr