ADMINISTRATION OF BONE MARROW CELLS INTO SURGICALLYINDUCED FIBROCOLLAGENOUS TUNNELS INDUCESANGIOGENESIS IN ISCHEMIC RAT HINDLIMB MODEL

LUIS PADILLA, M.D.,1�3* EDGAR KROTZSCH, Ph.D.,2

PAUL SCHALCH, M.D.,1 SIEGFRIED FIGUEROA, M.D.,1

ADRIANA MIRANDA, M.D.,2 ENRIQUE ROJAS, M.D., Ph.D.,2

SANDRO ESPERANTE, M.D.,1 FERNANDO VILLEGAS, M.D.,3

ANABEL S. DE LA GARZA, M.D.,3 AND

MAURICIO DI SILVIO, M.D.1

We established a comparative model of angiogenic inductionin previously formed fibrocollagenous tunnels in rat innerthigh muscles. A unilateral hindlimb chronic ischemia modelwas performed in male Sprague-Dawley rats. A device wasthen inserted in the central portion of the inner thigh muscles.Vascularity in the ischemic limb was determined by means ofan angiographic score, capillary/fiber ratio, and endothelialproliferation by histochemistry and immunohistochemistry.Autologous transplant of bone marrow, vascular endothelialgrowth factor (VEGF), or collagen-polyvinylpyrrolidone plusheparin induced significant vascularization of the ischemichindlimb when compared to saline solution. However, the

bone marrow group presented a higher angiographic scorethan the other two. No differences among groups were ob-served in capillary/fiber ratio or proliferation, except for theVEGF group, where capillary proliferating cells were signifi-cantly higher than in controls. Based on these results, bonemarrow-derived progenitor cells may constitute a safe andviable alternative for the induction of therapeutic angiogen-esis.

ª 2003 Wiley-Liss, Inc.

MICROSURGERY 23:568–574 2003

With the advancement of microsurgical techniques andthe saphenous vein technique in situ, it is now possibleto perform arterial revascularization procedures in moredistal locations for the management of leg ischemia.1�3

Rojas and Cervantes reported salvage in 82% of 47patients with distal arterial bypasses.4

In 1995, Rodriguez et al.5 reported the results of 26distal arterial reconstructions using infrapoplitealsaphenous vein short segments: 8 and 6 toward theposterior and the anterior tibial arteries, respectively; 5

to the peroneous artery; 4 to the pedial artery; and 3derived sequentially, for an overall 77% salvage rate. Sixof the 26 operated patients required a major amputationlater on.5

Therapeutic angiogenesis constitutes a new approachfor the management of critical limb ischemia, a conceptoriginated from the experimental work of Li-Qun et al.6

Baffour et al.7 Bauters et al.8 Takeshita et al.9 and Isneret al.10

Baumgartner et al.11 published the results of genetherapy in 10 patients with critical inferior limb ische-mia. These patients received intramuscular injections ofcDNA encoding a vascular endothelial growth factor(VEGF) isoform expression vector capable of replicat-ing in situ. Seven of 10 patients developed new collateralcirculation and thus were spared an amputation proce-dure.

In 2001, Epstein et al.12 reported a series of potentialadverse effects related to gene therapy and the use ofrecombinant proteins and growth factors. Among these,the most remarkable was the development of malignanttumors, as well as the proliferation of retinal and syn-ovial vessels, fragmentation of artherosclerotic plaques,and edema due to increased permeability. Up until now,there has only been one reported death related to the

1Department of Microsurgery and Experimental Surgery, Centro MedicoNacional ‘‘20 de Noviembre,’’ I.S.S.S.T.E., Mexico City, Mexico

2Connective Tissue Laboratory, Biomedical Research Division, Centro Med-ico Nacional ‘‘20 de Noviembre,’’ I.S.S.S.T.E., Mexico City, Mexico

3Microsurgery Unit, Department of Surgery, Facultad de Medicina, U.N.A.M.,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 03740 D.F., Mexico. E-mail: lpadilla@issste.gob.mx

Received 1 May 2003;3 Accepted 29 October 2003

Published online in Wiley InterScience (www.interscience.wiley.com). DOI:10.1002/micr.10208

ª 2003 Wiley-Liss, Inc.

administration of adenoviral vectors coding for angio-genic growth factors.

Asahara et al.13 and Shi et al.14 focused on thecapability of endothelial precursor cells to secrete VEGFand bFGF. Hamano et al.15 was able to induce angio-genesis after autologous bone marrow transplants in arat corneal model. These cells secrete bFGF and VEGFwith higher intensity when exposed to hypoxic condi-tions in culture.

Data published by our group demonstrate that theangiogenic process can be induced with alternativetechniques, e.g., the implantation of a synthetic pros-thetic material such as Silastic�, which generates fibro-collagenous tunnels that have an external vascularsupply.16 These neoformed tunnels may serve as a matrixfor supporting transplanted endothelial cells with thepurpose of inducing collateral vessel formation. Anothervery effective stimulator of angiogenesis is collagen-polyvinylpyrrolidone (collagen-PVP), combined withheparin. This mixture, administered in previouslyformed fibrocollagenous tunnels, was demonstrated toinduce and enhance angiogenesis in vivo.17

By means of a previously standardized microsurgicalmodel of chronic ischemia in the rat hindlimb,17 our aimwas to compare the development of collateral circula-tion induced by VEGF, collagen-PVP plus heparin, andbone marrow-derived pluripotential cells transplanted inneoformed fibrocollagenous tunnels.

MATERIALS AND METHODS

Male Sprague-Dawley rats weighing 300�350 g(Harland Tekland, Madison, WI) were fed ad libitumwith standard rodent chow (Nutrition International,Brentwood, MO) and treated in accordance with insti-tutional animal care guidelines from the Ethics andResearch Committee of the C.M.N. ‘‘20 de Noviembre,’’I.S.S.S.T.E., the General Health Legislation on Labo-ratory Animals Research,18 and the guide for use oflaboratory animals of the National Institutes ofHealth.19

Surgical Technique

After performing a medial laparotomy, the externaliliac artery was dissected free and ligated, preservingthe internal iliac artery. One week later, the femoralsegment was also dissected free, and after performing aproximal and distal ligation, it was excised. During thissecond procedure, a device consisting of an injectionport connected to three Silastic� tubes with an outsidediameter of 0.94 mm, 7 cm in length (Dow Corning,Midland, MI), was placed under the abdominal wall,

and a 4-cm segment of the three tubes, with threeholes, was inserted through the inner thigh muscles, inorder to induce the formation of fibrocollagenoustunnels. The distal ends of the tubes were fixed bynylon 3-0 to the surrounding muscle in order to avoiddisplacement. (Fig. 1). Prophylactic ampicillin (15 mgq.d.) was administered after each procedure for 3consecutive days.

One week after insertion of the injection devices,angiogenic factors were administered in a volume of200 ll, followed by 800 ll of saline solution to ensuretheir diffusion into the inner thigh muscles. Rats re-ceiving the bone marrow transplant underwent surgicalexposure of the left humerus with the aid of amicrodrill. A 2-mm perforation was performed, andbone marrow was aspirated using a heparinized sy-ringe attached to a catheter. An approximate volumeof 300 ll bone marrow aspirate was obtained fromeach rat.

Figure 1. Fibrocollagenous tunnel formation and angiogenic factor-

delivering device. Three Silastic� tubes are inserted through inner

thigh muscles of rat. Upper part of device is placed subcutaneously,

above abdominal wall muscles in right lateral position.

Bone Marrow-Induced Angiogenesis in Hindlimb 569

Angiogenic Factor Preparation

Red cells were lysed with 1 ml Tris-ammoniumchloride buffer (pH 7.2) for 10 min. After sequentialcentrifugations, the supernatant was eliminated, and thecell concentrate was resuspended in RPMI with 10%bovine fetal serum.15 Cell viability was determined bymeans of Trypan blue exclusion dye. Total cells (rangebetween 9.0 · 106 and 1.8 · 107 with over 95% viability)were administered to each rat, as previously described.

Collagen-PVP (Fibroquel�, Aspid S.A. de C.V.,Mexico City, Mexico) was mixed 1:1 with heparin 1,000IU prior to being injected into the device.

Four micrograms of rhVEGF (R&D Systems, Min-neapolis, MN) were injected into the device as described(Table 1).

Device Extraction

A small incision on the skin was performed 1 weekafter a single administration of the angiogenic factors,and the injection devices were removed by gentle traction.

Angiographic Studies

Four weeks after administration of angiogenic fac-tors, a contrasted angiography was performed by can-nulating the abdominal aorta above the bifurcation ofthe iliac arteries. The arteries were maintained permeablewith 10 ml of a heparin/lidocaine solution. Three milli-liters of contrast medium (Optiray-30, Mallinckrodt,Quebec, Canada) were injected under fluoroscopy, andan X-ray image of both hindlimbs was obtained. Ablinded observer placed a transparent acetate over the X-ray image and copied the vascular pattern with ink. Thenumber of intersections was counted using a millimeterscaled paper in an area of 2.5 · 2.5 cm, as previouslydescribed (angiographic score).8

Histochemistry, Immunohistochemistry, and

Electron Scanning Imaging

One biopsy from the central portion of the innerthigh muscles was obtained from both limbs after ang-iography; the contralateral served as control. For hist-

ochemical and immunohistochemical studies, sampleswere embedded in tissue-freezing medium and snap-frozen in liquid nitrogen. Samples were then maintainedat )70�C until processing. Serial 6-lm-thick cryocutswere obtained and stained both for alkaline phosphatase(AP) activity20 in order to detect microvasculature, andfor detection of proliferating cell nuclear antigen(PCNA). Capillary/fiber ratio was determined bycounting capillaries and muscle fibers at ·400 in threerandom linearly aligned fields in the central portion ofthe sample in two serial sections.

In order to determine the presence of proliferatingcells, sections were treated by a heat-induced epitoperetrieval technique with citrate buffer solution, accord-ing to the manufacturer’s instructions (Zymed, SanFrancisco, CA). An immunohistochemical analysis ofPCNA21 was performed with a Zymed� PCNA stainingkit (Clone PC-10, Zymed, San Francisco, CA). Prolif-erating cells were quantitated by the percentage of im-munoreactive nuclei determined at ·400 in three randomlinearly aligned fields in the central portion of the sec-tion in two serial sections per specimen. Both assays,histochemistry and immunohistochemistry, were per-formed in a blinded form by two independent observers.

Scanning Electron Microscopy Technique

Following fixation in glutaraldehyde, dissected fi-brocollagenous tunnels were dehydrated in a 10�100%alcohol gradient and inmersed in acetone. Tissues weretransported to a critical point (Sandri 780) and placedand adhered with silver stain in a 1-cm-diameter, 1.5-cm-high object-carrier. Mounted specimens were cov-ered in gold at 40 atm vaccum for 3 min and observed ina Jeol JSM 5200 microscope.

Statistical Analysis

Angiographic scores were analyzed with the TukeyHSD test, while percentages of PCNA immunoreactivenuclei were analyzed by multiple-comparison LSD test.Statistical significance was set at P £ 0.05.

RESULTS

Angiographic Analysis

The number of vascular intersections (Fig. 2), as aparameter of venous and arterial reconnection, was re-corded and expressed as the mean angiographic score(MAS) (Fig. 3).8 MAS for the bone marrow cells-treatedgroup (37.5 ± 2.3) was significantly higher than theVEGF, collagen-PVP + heparin, or saline solution-

Table 1. Groups Table4

Group n Treatment

I 10 SalineII 7 Vascular endothelial growth factor (VEGF)III 10 Collagen-polyvinylpyrrolidone plus

heparin (1:1)IV 10 Bone marrow (red cells removed)

570 Padilla et al.

Figure 2. Vascular pattern obtained from angiographic images. Area of 2.5 · 2.5 cm in a millimeter-scaled paper is used to calculate number

of intersections, which is expressed as angiographic score. a: Saline. b: VEGF. c: Collagen-PVP + heparin. d: Bone marrow.

Bone Marrow-Induced Angiogenesis in Hindlimb 571

treated groups (26.7 ± 2.1, 25.2 ± 3.9, and 14.6 ± 0.8,with P £ 0.05, 0.008, and 0.0002, bone marrow vs. othergroups; respectively). There were no significant differ-ences between the VEGF and collagen-PVP + heparingroups, but both groups had a significantly higher an-giographic score compared to the saline-injected controlgroup (P £ 0.02 and 0.03, respectively).

On the other hand, a linear correlation for the an-giographic score was observed for the number oftransplanted cells in the bone marrow group (r =0.99178), where the highest number of transplanted cells(1.827 · 107 cells) showed a 2-fold increase in MAS,when compared to the VEGF or collagen-PVP + hep-arin groups, and a 3.5-fold increase when compared tothe control group (Fig. 4).

Histochemical, Immunohistochemical, and

Ultrastructural Findings

Microvasculature, as well as proliferating cells, wereanalyzed in the central portion of the inner thigh mus-cles by means of histochemical and immunohistochem-ical methods. In contrast to angiographic data, therewere no statistically significant differences in regard tocapillary/fiber ratios among groups. However, the per-centage of proliferating endothelial cells (evidenced bymarked nuclei in morphologically identified endotheli-um and alkaline phosphatase-positive regions) was sig-nificantly higher only in the VEGF-treated group (15.1± 4.3, P < 0.03 vs. controls), while the other groups

presented PCNA levels similar to or slightly higher thanthe contralateral limb (6.7 ± 1.3; saline 10.8 ± 1.9;

1collagen-PVP + heparin = 8.3 ± 2.4; bone marrow =12.9 ± 2.6; Fig. 5).

Interestingly, we also found a specific positive im-munoreaction inside the muscular fiber, perhaps sec-ondary to marked myocyte nuclei. These marks werenot detected in the negative control slide (Fig. 6).

Electron scanning microscopy of a representativecross section of fibrocollagenous tunnels and peripheralvascular invasion, after administration of VEGF, col-lagen-PVP + heparin, or bone marrow, showed tunnelssurrounded by fibrous tissue and smooth muscle, with-out inflammatory infiltrate and scarce endothelial in-vasion (Fig. 7).

DISCUSSION

Our data show that bone marrow cells, VEGF, orcollagen-PVP plus heparin when transplanted into pre-viously formed fibrocollagenous tunnels in the ischemichindlimb of the rat are capable of inducing collateralvessel formation (Fig. 7). Bone marrow has a strongereffect, followed by VEGF and collagen-PVP + heparin;all three of them were significantly stronger whencompared to saline-injected controls (Fig. 3).

Previous work showed that the mixture of collagen-PVP plus heparin administered into fibrocollagenoustunnels16,17 increased vessel formation. However, the re-sults presented here had significantly better effects whenautologous bone marrow cells were injected in the tunnel.

The device designed for the administration of angi-ogenic factors served the dual purpose of inducing theformation of fibrocollagenous tunnels with externalvascular supply (thus creating an internal smooth sur-face covered by collagen, which is an excellent structural

Figure 3. Angiographic score. Bars represent mean ± SEM for each

treatment group. I, saline (n = 10); II, VEGF (n = 7); III, collagen-PVP

+ heparin (n = 10); and IV, bone marrow (n = 10). *Group IV vs. I, II,

and III, P £ 0.0002, 0.008, and 0.05, respectively. �Groups II and III

vs. I, P £ 0.02 and 0.03, respectively, by means of Tukey HSD test.

Figure 4. Linear correlation of angiographic score vs. total number

of transplanted bone marrow cells. Circles represent number of bone

marrow cells transplanted and resulting number of intersections.

Correlation coefficient r = 0.9918; dashed lines represent 95% con-

fidence interval.

572 Padilla et al.

support for endothelial cells),17 and of facilitating theadministration of angiogenic factors into the ischemicmuscle.

Histochemical and immunohistochemical analysesdid not show significant differences among differenttreatment groups, with the exception of the VEGF-treated group, which showed an increase in PCNA ex-pression compared to nonischemic controls (Fig. 5).Still, the proportion of the microvasculature and the

proliferating PCNA-expressing cells did not reach themagnitude reported in the literature.21 A possible ex-planation for this is that our model of ischemia involvesprogressive steps, first by ligating of the external iliacartery and, a week later, the femoral segment. This se-quential impairment of perfusion creates chronic ische-mia.21 Greater-diameter vessels, present before theischemic process, as well as smaller-diameter vessels thatsuffered changes at the beginning of ischemia, wouldhave, by the time the biopsies were taken (4 weeks afterinjection), assumed their final anatomical and histolog-ical appearance as a consequence of proliferation andremodeling processes. The elapsed time between the lastsurgical procedure and the harvesting of muscle samples(4 weeks) was probably enough to allow collateral bloodvessels to form from vessels of greater diameter (col-lateralization), present according to our angiographicfindings. This could explain why there were no impor-tant microscopic differences among groups.

Even though the administration of angiogenic me-diators occurred as a one-time event and the adminis-tered dose of VEGF was 20 times less than the standardexperimental dose reported in the literature, we didobserve a significant increase of proliferating capillariesin the central portion of the ischemic muscle at themicroscopic level. While there are many variants toangiogenic therapy, most of them based on the admin-istration of growth factors, either directly or via a-denoviral vectors, our study opens the possibility ofcombined approaches using prosthetic devices thatstimulate the formation of a kind of scaffolding whereprecursor cells can be transplanted. These cells differ-entiate into endothelial cells that ultimately occupy thesmooth, collagen-lined lumen of the fibrocollagenous

Figure 5. Proliferating cells were assessed quantitatively at ·400magnification by calculating percentage of immunoreactive nuclei.

Bars represent mean ± SEM for each group of treatment. Healthy

limb refers to normal contralateral limb. I, saline solution; II, VEGF;

III, collagen-PVP + heparin; and IV, bone marrow. *Versus healthy

limb, P £ 0.03, by means of LSD test.

Figure 6. Immunohistochemical staining of PCNA in central portion

of inner thigh muscles. Proliferating cells (marked nuclei, arrows)

were significantly higher only in VEGF-treated group (c). a: Contra-

lateral limb. b�e: Representative microscopic fields of ischemic

limbs injected with saline, VEGF, collagen-PVP + heparin, and bone

marrow, respectively. f: Negative control. g: PCNA-positive control

(rat intestine) (·200 magnification). [Color figure can be viewed in the

online issue, which is available at www.interscience.wiley.com.]

Figure 7. Electron scanning micrograph showing cross section of

fibrocollagenous tunnel surrounded by extracellular matrix and em-

bedded small vessels. Scale represents 500 lm.

Bone Marrow-Induced Angiogenesis in Hindlimb 573

tunnel and also secrete angiogenic growth factors inresponse to the hypoxic condition of the surroundingtissues.15,22 Both mechanisms (cell differentiation in situand the production of cytokines and angiogenic growthfactors) could account for the linear correlation betweentotal number of cells transplanted and the MAS foundin the bone marrow-treated group. It is worth notingthat the injection of collagen-PVP plus heparin, whilenot as potent as the other mediators studied in this ex-periment, did cause a significant increase of vascularintersections, which correlates with previous reports,17

and allowed for partial recovery from ischemia. Futureexperiments could include a combination of bone mar-row transplant and collagen-PVP + heparin in order tostudy possible synergistic effects.

Finally, it is important to highlight the fact that whilehistological analyses focused on the presence and pro-liferation of capillaries, we were also able to observe avery important presence of PCNA immunoreactivity inthe intrafibrillary space of the muscle. We were able todetermine that both collagen-PVP + heparin and bonemarrow stimulated the highest expression of PCNA.There is no clear mechanism for such an effect, althoughit could be inferred as the result of direct stimulation ofmuscle proliferation or an indicator of tissue repair, evenif the observed immunoreactivity does not necessarilycorrelate with the angiographic findings (Fig. 6, arrow).

This work demonstrated an improvement in the in-duction of vessel formation when compared to previousstudies from our group.4,5,16,17 It also encourages thedevelopment of new surgical models, such as a chronicischemic hindlimb model in dogs, in order to measurethe effect of the administration of endothelial precursorssuch as CD34+ cells after induction of looped fibro-collagenous tunnels.

ACKNOWLEDGMENTS

The authors thank mathematician Jorge Galicia forhis advice on statistical analysis.

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