autologous bone marrow cells transplantation in patients with advanced critical limb ischemia: no...

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Cell Transplantation, Vol. 21, pp. 1909–1918, 2012 0963-6897/12 $90.00 + .00Printed in the USA. All rights reserved. DOI: http://dx.doi.org/10.3727/096368912X636948Copyright 2012 Cognizant Comm. Corp. E-ISSN 1555-3892 www.cognizantcommunication.com

Received June 13, 2011; final acceptance October 22, 2011. Online prepub date: April 2, 2012.Address correspondence to Juraj Madaric, M.D., Ph.D., Department of Cardiology and Angiology, National Cardiovascular Institute, Pod Krasnou horkou 1, 833 48 Bratislava, Slovakia. Tel: 00421259320276; E-mail: [email protected]

No Difference in Intra-Arterial and Intramuscular Delivery

of Autologous Bone Marrow Cells in Patients With

Advanced Critical Limb Ischemia

Andrej Klepanec,*† Martin Mistrik,‡ Cestmir Altaner,§ Martina Valachovicova,* Ingrid Olejarova,† Roman Slysko,† Tibor Balazs,† Terezia Urlandova,† Daniela Hladikova,† Branislav Liska,†

Jan Tomka,† Ivan Vulev,*† and Juraj Madaric*†

*Slovak Medical University, Bratislava, Slovakia

†National Cardiovascular Institute, Bratislava, Slovakia

‡Clinic of Haematology and Transfusiology, Faculty Hospital, Bratislava, Slovakia

§Institute of Experimental Oncology, Slovak Academy of Science, Bratislava, Slovakia

Stem cell therapy has been proposed to be an alternative therapy in patients with critical limb ischemia (CLI), not eligible for endovascular or surgical revascularization. We compared the therapeutic effects of intramuscular (IM) and intra-arterial (IA) delivery of bone marrow cells (BMCs) and investigated the factors associated with thera-peutic benefits. Forty-one patients (mean age, 66 ± 10 years; 35 males) with advanced CLI (Rutherford category, 5 and 6) not eligible for revascularization were randomized to treatment with 40 ml BMCs using local IM (n = 21) or selective IA infusion (n = 20). Primary endpoints were limb salvage and wound healing. Secondary endpoints were changes in transcutaneous oxygen pressure (tcpO

2), quality-of-life questionnaire (EQ5D), ankle–brachial

index (ABI), and pain scale (0–10). Patients with limb salvage and wound healing were considered to be respond-ers to BMC therapy. At 6-month follow-up, overall limb salvage was 73% (27/37) and 10 subjects underwent major amputation. Four patients died unrelated to stem cell therapy. There was significant improvement in tcpO

2

(15 ± 10 to 29 ± 13 mmHg, p < 0.001), pain scale (4.4 ± 2.6 to 0.9 ± 1.4, p < 0.001), and EQ5D (51 ± 15 to 70 ± 13, p < 0.001) and a significant decrease in the Rutherford category of CLI (5.0 ± 0.2 to 4.3 ± 1.6, p < 0.01). There were no differences among functional parameters in patients undergoing IM versus IA delivery. Responders (n = 27) were characterized by higher CD34+ cell counts in the bone marrow concentrate (CD34+ 29 ± 15×106 vs. 17 ± 12×106, p < 0.05) despite a similar number of total nucleated cells (4.3 ± 1.4×109 vs. 4.1 ± 1.2×109, p = 0.66) and by a lower level of C-reactive protein (18 ± 28 vs. 100 ± 96 mg/L, p < 0.05) as well as serum leukocytes (8.3 ± 2.1×109/L vs. 12.3 ± 4.5×109/L, p < 0.05) as compared with nonresponders (10 patients). Both IM and IA delivery of autologous stem cells are effective therapeutic strategies in patients with CLI. A higher concentration of CD34+ cells and a lower degree of inflammation are associated with better clinical therapeutic responses.

Key words: Autologous stem cells; Critical limb ischemia; Angiogenesis; Intramuscular delivery; Intra-arterial delivery

Several preclinical and clinical studies showed that

delivery of autologous bone marrow cells (BMCs) can

improve blood circulation and tissue perfusion and

thus prevent amputation via the induction of collat-

eral and capillary growth in a process called “thera-

peutic angiogenesis” (2,5,12,18,22,31,35). However,

the optimal route of administration of cells remains

unclear. Intra-arterial (IA) as well as intramuscular

(IM) delivery methods have shown promising results

in promoting neoangiogenesis (2,5,12,35), although

direct comparison between both routes of BMC deli-

v ery is lacking. Accordingly, we conducted a randomized

INTRODUCTION

Critical limb ischemia (CLI) is the end stage of periph-

eral arterial disease (PAD) characterized by ischemic rest

pain, ulcers, or gangrene, with a significant risk of loss

of the affected limb. The therapeutic options for patients

with failed endovascular or surgical revascularization or

in those in which these procedures cannot be carried out

(20–30% of CLI patients) are very limited. About 40%

of these high-risk patients will require amputation within

6 months of the initial diagnosis, whereas 20% will die

(23). The quality of life (QoL) of these patients is poor

and comparable to those with terminal cancer (1).

1910 KLEPANEC ET AL.

clinical study to compare IM versus IA delivery of autol-

ogous BMCs in “no-options” patients with advanced CLI.

To understand the underlying mechanism of the thera-

peutic effects of stem cells, we sought to address fac-

tors associated with therapeutic benefit in response to

cell therapy.

MATERIALS AND METHODS

Patients

Between October 2009 and August 2010, 41 patients

(mean age, 66 ± 10 years; 35 males) with advanced CLI

(Rutherford category, 5 or 6) after failed or impossible

revascularization were randomized to application of 40 ml

of bone marrow concentrate via the local IM route (n = 21)

or via selective IA infusion (n = 20).

Inclusion Criteria. (1) Patients over 18 years of age

with ischemic skin lesions (ulcers or gangrene) with

a CLI Rutherford category of 5 or 6 according to the

TransAtlantic InterSociety Consensus (TASC) classifica-

tion (minor or major tissue loss) (23). (2) CLI defined

by ankle–brachial index ≤ 0.4, or ankle systolic pressure

< 50 mmHg, or toe systolic pressure < 30 mmHg, or tran-

scutaneous oxygen pressure (tcpO2) < 30 mmHg. (3) No

option for endovascular or surgical revascularization

assessed by a vascular surgeon and interventionalist. (4)

Failed revascularization defined as no change of clinical

status with the best standard care 4 weeks after endovas-

cular or surgical revascularization.

Exclusion Criteria. (1) Life expectancy < 6 months.

(2) Evidence of malignancy during last 5 years. (3)

Proliferative retinopathy. (4) Critical coronary artery dis-

ease or unstable angina pectoris. (5) End-stage kidney

disease and patient on dialysis. (6) Bone marrow disease

(e.g., myelodysplastic syndrome, severe anemia, leuko-

penia, and thrombocytopenia).

Method of BMC Isolation. Isolation of stem cells

was undertaken under analgosedation with propofol. A

total of 240 ml of bone marrow from both posterior iliac

crests was harvested using a standard disposable needle

for bone marrow aspiration. Bone marrow aspirate was

processed using a SmartPreP2 Bone Marrow Aspirate

Concentrate System (Harvest, Plymouth, MA, USA),

which uses gradient density centrifugation to provide

40 ml of bone marrow-rich product for all blood ele-

ments within 15 min. The biological potential of this

system has been evaluated in a mouse model of hind-

limb ischemia with similar or greater functional activ-

ity compared with the Ficoll isolation procedure as the

current “gold standard” (10) and has also been tested in

several trials in patients with CLI (2,25).

Administration of Stem Cells. Immediately after the

harvesting and centrifugation of stem cells, BMCs were

administered by IM (group A) or IA (group B) methods.

In group A, 40 ml of BMCs were administered under anal-

gosedation with propofol by deep injections with a 23-G

needle into the muscles of the affected limb along the

crural arteries, with each injection being ~1 ml. In group

B, IA injection of 40 ml of BMCs was undertaken from

a percutaneous retrograde contralateral femoral approach

or antegrade femoral approach under local anesthesia at

the site of arterial occlusion of the affected limb using a

4-F catheter at 800 ml/h. The duration of procedures in

both groups was ~1 h.

Preprocedure Assessment and Follow-up. All patients

were examined before, 90 days, and 6 months after BMC

delivery. Peripheral blood tests such as blood count and

basal serological parameters (including C-reactive pro-

tein, CRP) were assessed. The total concentration of

mononuclear cells (BMMCs/MNCs) and CD34+ cells in

bone marrow concentrates was evaluated.

Digital subtraction angiography (DSA) was under-

taken 1 day before transplantation and 6 months after

transplantation with strictly fixed parameters of the

amount of contrast medium, constant speed of injec-

tion of contrast medium, catheter size, and position

of catheter tip. Two experienced operators evaluated

the development of new vessels in a blinded fashion

by semiquantitative analyses as reported elsewhere (31).

New collateral vessels were assessed as +0 (no develop-

ment of collateral vessels), +1 (slight), +2 (moderate),

or +3 (rich).

Measurement of the resting ankle–brachial index (ABI)

was done according to validated standards (27). It was cal-

culated as the quotient of the highest ankle pressure and

highest brachial systolic blood pressure (normal values,

0.95–1.2). Transcutaneous oxygen pressure (tcpO2) of the

affected limb was assessed using a TCM400 Mk2 moni-

tor (Radiometer Medical ApS, Copenhagen, Denmark).

TcpO2 was measured at the forefoot in the supine position

with an electrode temperature of 44°C. Wound character-

istics were documented by digital photography. Wound

healing was evaluated by two independent physicians.

Pain scale was measured with a visual analog scale (VAS)

graded form 0 to 10.

Patients were discharged the day after the procedure

on dual antiplatelet therapy (aspirin and clopidogrel) and

statin therapy. All patients received conventional wound

care during follow-up.

QoL before, 3 months, and 6 months after BMC appli-

cation was assessed by the EuroQol questionnaire (6).

Using a VAS, patients rated their overall health status

from 0 (“worst”) to 100 (“best”) imaginable health.

Endpoints. The primary endpoint was limb salvage and

improvement in wound healing within 6-month follow-up.

Patients with limb salvage and wound healing were con-

sidered to be responders to BMC therapy. Secondary

STEM CELL DELIVERY IN CRITICAL LIMB ISCHEMIA 1911

endpoints were change in tcpO2, Rutherford category,

QoL, and pain VAS after BMC transplantation.

The study design was approved by the local ethical

committee of National Cardiovascular Institute, Bratislava.

All included patients were informed about the nature of the

study and gave their written informed consent.

Statistical Analyses

Data evaluation was undertaken using a statistical

software package SPSS 13.0 (SPSS Inc., Chicago, IL,

USA). Discrete variables are presented as counts and

percentages. Continuous variables are presented as mean

values ± SD. Gaussian distributions of data were tested

by the Kolmogorov–Smirnov test. The paired t test was

used to compare values before and after BMC trans-

plantation. The frequencies of categorical variables

were compared using Fisher‘s exact test. Mean values

for continuous parameters were compared using the

Student‘s t test and Mann–Whitney test as appropriate.

Multivariate logistic regression analysis (binary logis-

tic regression) was used to study predictors of clinical

benefit after BMC application. For all analyses, p < 0.05

was considered significant.

RESULTS

Baseline Characteristics and Overall Results

The baseline characteristics of both groups are given

in Table 1. In all 41 patients, the underlying cause of

obstructive arterial disease was atherosclerosis. During

follow-up, four patients died (two due to heart failure,

one due to myocardial infarction, and one as a result of

pneumonia). At 6-month follow-up, the combined pri-

mary endpoint of limb salvage and wound healing was

met in 27 of the surviving 37 patients (73%). However,

in 10 patients, major amputation was required due to CLI

progression. Importantly, most cases of amputation (7/10)

happened during the first month after BMC delivery.

Table 2 shows the functional results of BMC appli-

cation in patients with limb salvage at 6 months in all

patients and that in the IA group and IM group. There was

significant improvement in wound healing, tcpO2, pain

scoring, and QoL, as well as in CLI Rutherford category

at 3-month follow-up, and the results were sustained or

even more pronounced at 6 months (Figs. 1 and 2).

DSA did not reveal detectable development of new

collateral vessels after 6 months compared with baseline

angiograms (grades 0 to +3: 0.21 ± 0.43) (Fig. 3).

Intramuscular Versus Intra-Arterial Application

Table 3 shows a comparison of functional outcomes

after IM and IA delivery of BMCs in patients with limb

salvage. Both procedures were well tolerated without

periprocedural complications. There were no differences

in IM versus IA application in either endpoint. The preva-

lence of limb salvage at 6 months was 72% in the IM

group compared with 74% in the IA group ( p = 0.94).

Similarly, wound healing was observed in 13 patients

(72%) in the IM group compared with 14 patients (74%)

Table 1. Baseline Characteristics of Patients

All Patients

(n = 41)

Group A

(IM, n = 21)

Group B

(IA, n = 20)

p

(IM vs. IA)

Age (years) 66 ± 10 66 ± 10 66 ± 11 0.86

Sex (males) 35 (85%) 17 (81%) 18 (90%) 0.41

Diabetes mellitus 28 (68%) 15 (71%) 13 (65%) 0.67

Arterial hypertension 33 (80%) 17 (81%) 16 (80%) 0.62

Hyperlipidemia 21 (51%) 13 (62%) 8 (40%) 0.96

BMI 28 ± 4 29 ± 4 26 ± 4 0.06

LVEF (%) 56 ± 8 60 ± 9 55 ± 7 0.43

Smoking 17 (41%) 9 (43%) 8 (40%) 0.85

Rutherford category 5.0 ± 0.2 5.0 5.1 ± 0.3 0.16

Creatinine (µmol/L) 94 ± 49 99 ± 66 88 ± 19 0.47

CRP (mg/L) 38 ± 60 44 ± 73 31 ± 42 0.50

Leu (109/L) 9.2 ± 3.3 9.2 ± 3.3 9.2 ± 3.5 0.99

Fbg (g/L) 4.1 ± 0.9 3.9 ± 1.0 4.3 ± 0.7 0.11

BMMC count (109 cells) 4.2 ± 1.4 4.4 ± 1.5 4.0 ± 1.3 0.45

CD34+ count (106 cells) 26 ± 14 28 ± 17 23 ± 11 0.30

Previous PTA/surgery 29 (71%) 14 (67%) 15 (75%) 0.73

Previous CABG/PCI 6 (15%) 3 (14%) 3 (15%) 1.0

Post-MI 13 (32%) 6 (29%) 7 (35%) 0.74

BMI, body mass index; BMMC, bone marrow mononuclear cells; CABG, coronary artery bypass grafting; CRP, C-reactive protein; Fbg, fibrinogen; IA, intra-arterial; IM, intramuscular; Leu, leukocyte level in peripheral blood; LVEF, left ventriclar ejection fraction; MI, myocardial infarction; PCI, percutaneous coronary intervention.


Figure 1. Nonhealing ulcers before and 6 months after intramuscular (A) and intra-arterial (B) deliv-ery of BMCs. BMCs, bone marrow cells; tcpO

2, transcutaneous oxygen pressure.

Table 2. Outcomes of BMC Delivery After 6 Months in Patients With Limb Salvage

Overall Group (n = 27) Group A (IM, n = 13) Group B (IA, n = 14)

Base 6 Months p Base 6 Months p Base 6 Months p

tcpO2 (mmHg) 15 ± 10 29 ± 13 <0.001 13 ± 10 30 ± 12 <0.01 15 ± 11 28 ± 14 <0.01

ABIa 0.9 ± 0.4 0.9 ± 0.2 0.88 0.9 ± 0.4 0.9 ± 0.2 0.34 0.9 ± 0.4 0.9 ± 0.2 0.22

Pain scale (0–10) 4.4 ± 2.6 0.9 ± 1.4 <0.001 3.8 ± 2.5 0.6 ± 1.0 <0.001 4.8 ± 2.6 0.8 ± 1.3 <0.001

QoL (0–100) 51 ± 15 70 ± 13 <0.001 55 ± 17 69 ± 12 <0.05 49 ± 14 72 ± 13 <0.01

Rutherford category

(0–6)

5.0 ± 0.2 4.3 ± 1.6 <0.01 5.0 3.6 ± 1.4 <0.01 5.1 ± 0.3 3.7 ± 1.4 <0.01

Wound size (cm2) 8.2 ± 6.8 2.8 ± 6.3 <0.001 7.1 ± 4.8 2.3 ± 2.1 <0.001 8.9 ± 8.1 3.2 ± 8.1 <0.001

ABI, ankle–brachial index; BMC, bone marrow cells; QoL, quality-of-life; tcpO2, transcutaneous oxygen pressure; IA, intra-arterial; IM, intramus-

cular; p = 6 months versus baselineaMediasclerosis was noted in 22/27 patients (81%).


Figure 2. Improvement in trancutaneous oxygen pressure and pain scale 3 and 6 months after BMC delivery: IM versus IA group. BMC, bone marrow cells; IA, intra-arterial; IM, intramuscular; tcpO

2, transcutaneous oxygen pressure. *p = NS IM versus IA; †p < 0.005 baseline

versus 6 months.

in the IA group (p = 0.94). There were no procedure-

related complications in either group.

Responders Versus Nonresponders to Cell Therapy

Table 4 shows the characteristics of responders ver-

sus nonresponders to BMC therapy. Responders (n = 27)

were characterized by a higher CD34+ cell count in the

BMC product (29 ± 15 × 106 vs. 17 ± 12 × 106, p < 0.05)

despite having a similar number of total nucleated cells

(4.3 ± 1.4 × 109 vs. 4.1 ± 1.2 × 109, p = 0.66). Responders

had lower CRP levels (18 ± 28 vs. 100 ± 96 mg/L, p < 0.05)

and peripheral blood leukocyte count (8.3 ± 2.1 × 109/L

vs. 12.3 ± 4.5 × 109/L, p < 0.05) as compared with nonre-

sponders (10 patients).

Upon univariate analysis, a CD34+ cell count >20 × 106

was associated with a positive therapeutic response to

stem cell therapy [p = 0.015, odds ratio (OR) 4.7, 95%

confidence interval (CI) 1.15–19.24], and peripheral

blood leukocyte count >10 × 109/L was associated with

a negative therapeutic response (p = 0.006, OR 2.1,

95% CI 1.06–4.1), similar to the effect of CRP level

>10 mg/L ( p = 0.038, OR 1.54, 95% CI 1.01–2.32).

Upon multivariate analysis (binary logistic regression),

the number of administrated CD34+ cells >20 × 106

emerged as an independent predictor of clinical benefit

after BMC application ( p = 0.03). A peripheral leukocyte

count >10 × 109/L was shown to be an independent pre-

dictor of negative therapeutic response to cell therapy

( p = 0.048). According to the receiver operating charac-

teristic (ROC) analysis, a cutoff limit for CD34+ cells of

20 × 106 in delivered bone marrow concentrate was pre-

dictive for a positive clinical response with 80% specific-

ity and 65% sensitivity (Fig. 4).

DISCUSSION

The present study investigates the effects of two meth-

ods of delivery of autologous BMCs on the progression

of advanced CLI. The main findings can be summarized

as follows: (1) IM and IA delivery routes are effective and

comparable in inducing a therapeutic effect in CLI and (2)

higher CD34+ cell counts and a lower degree of inflam-

mation are associated with a better clinical response to

BMC administration.

Delivery of Autologous BMCs in Patients With CLI

Several authors have reported the clinical benefit of

administration of autologous BMCs in patients with CLI

(2,5,12,18,22,31,35). BMC delivery has been associated

with limb salvage, increase in tcpO2, blood flow perfu-

sion, or the ABI. The results of the present study cor-

roborate the positive effects of BMC therapy on wound

healing, tcpO2, pain scoring, Rutherford category, as

well as QoL. Conversely, the ABI did not differ after 6

months in the present study, similar to the findings in the

PROVASA trial (35). Of note, most of our patients suf-

fered from diabetes mellitus, with the high prevalence of

mediasclerosis [22/27 subjects (81%) in the responders

group], where noncompressible ankle arteries precluded

meaningful determination of the ABI. Importantly, the

prevalence of limb salvage 73% in the present study was

similar to that of other studies reporting the effects of cell

therapy at 6 months (15,37).

Intramuscular Versus Intra-Arterial Routes

of BMC Delivery

There is ongoing discussion about the optimal method

of cell delivery in CLI. IM as well as IA methods of


Figure 3. Digital subtraction angiography before and 6 months after BMC delivery: IM (A) versus IA (B) group.

administration of stem cells have shown promising results

in achieving therapeutic benefit (2,5,12,25,35).

One potential advantage of IM delivery is creation

of “local depots” of implanted cells with increased

local paracrine activity in the ischemic area. However,

the prevalence of cell retention and survival times after

IM injection into ischemic limb muscle is not known.

For IA delivery, the homing of administrated BMCs in

the zone of ischemia is crucial (4). With selective IA

delivery, stem cells can reach the border zone of maxi-

mum ischemia by blood flow, although the degree of

cell uptake from the circulation and from engraftment

is unknown.

In the clinical setting, the question of the optimal

route of BMC delivery has been addressed only in small

studies (8,32). Our results from direct head-to-head

comparison of different administration routes indicate

that IM as well as IA methods of BMC delivery are

effective in limb salvage and wound healing, with no

significant differences in various functional surrogate

endpoints between the techniques. Our clinical obser-

vations corroborate experimental findings of a similar

level of angiogenic activity after IM and IA injections in

the rat ischemic hind-limb model (36). Likewise, they

are in agreement with a recent study showing no differ-

ences in the extent of perfusion improvement after IM


administration or combined IM and IA administration in

patients with CLI (32).

There are various techniques of IM or IA administra-

tion of stem cells. For IA methods, slow delivery over

3 min upon injection into the superficial femoral artery

(19) or IA infusion at 900 ml/h (5) has been described.

We undertook selective IA delivery of BMCs at a con-

trolled rate of 800 ml/h at the site of arterial occlusion.

For IM administration, in most clinical trials, injection

has usually been done using a symmetric grid with a fixed

number of injections (2,25). In the present study, BMC

administration into the muscles of the affected limb was

carried out along the crural arteries.

Functional improvement after IM and IA adminis-

tration was observed in the absence of macrovascular

changes of visible collaterals or arteriogenesis of the

affected limb upon DSA. This finding supports the con-

cept that the therapeutic effects of BMCs in CLI are pri-

marily exerted at the microcirculation and that DSA is

not a suitable method for the evaluation of therapeutic

angiogenesis (33).

Predictors of Therapeutic Responses to Delivery of

Autologous BMCs

The potential predictors of the therapeutic response

to cell-based therapy in CLI have not been clearly eluci-

dated. In the present study, two factors were predictive of

the response to cell therapy. The number of administrated

CD34+ cells, but not the total number of nucleated cells

(bone marrow mononuclear cells, BMMCs), was strongly

related to clinical benefit. This result partially contradicts

recent findings in the PROVASA trial in which CD34+

cells and BMMC numbers were independent predictors of

improved ulcer healing (35). Conversely, they are consis-

tent with various studies demonstrating the superior effects

of enriched CD34+ cells as compared with BMMCs. In

fundamental studies, the surface expression of CD34,

CD133 and vascular endothelial growth factor receptor-2

[VEGFR-2/kinase insert domain receptor (KDR)] identi-

fied a population of endothelial progenitor cells (EPCs)

with enhanced potency for neovascularization of isch-

emic tissue (7,14,16,24). Likewise, CD34+ cells could

restore the microcirculation and improve tissue perfusion

in preclinical models (13,14) as well as in clinical series

(21,28) that appeared to be superior to BMMCs alone (13).

Furthermore, enriched CD133+ progenitor cells demon-

strated positive functional effects in patients with chronic

as well as recently infarcted myocardium (3,29). Evidence

that CD34+ cells may be pivotal for therapeutic benefits is

supported by the notion that mononuclear cells depleted

of CD34+ cells do not improve myocardial function in a

murine infarct model (16). Nevertheless, it is likely that

nonhematopoietic stem cells (mesenchymal or stromal

cells), with their high paracrine ability, could also contrib-

ute to the beneficial effect of cell therapy. Mesenchymal

cells, although found in low numbers in mononuclear cell

fractions, are a potent source of trophic cytokines and have

been shown to exert pro-angiogenic effects regardless of

bone marrow or adipose origin (9,17). In addition, the

composition of the bone marrow concentrate used in the

present study included a small portion of an erythrocyte

layer together with a high number of platelets. Platelets, as

a rich source of paracrine activity, have been shown to aug-

ment the formation of collateral vessels in ischemic tissue

in the presence of mononuclear cells (11).

Second, elevated inflammatory markers (e.g., leukocyte

count, CRP levels) were predictive of a negative thera-

peutic response to cell delivery. CRP is considered to be

one of the strongest predictors of vascular death (26) and

appears to be an important mediator of atherogenesis (30).

It can exert a harmful effect on EPC function, resulting in

impaired repair of vessels and impaired neovasculariza-

tion of ischemic tissues (34). Accordingly, a reduced CRP

level has been shown to be a major predictor of successful

outcome in percutaneous transluminal angioplasty in dia-

betic patients with infected foot ulcers (20). Our findings

Table 3. Six-month Comparison of Functional Outcomes of

BMC Delivery in Patients With Limb Salvage

Group A

(IM, n = 13)

Group B

(IA, n = 14)

p

(IM vs. IA)

∆ tcpO2 (mmHg) 13.4 ± 12.4 13.5 ± 13.4 0.66∆ tcpO

2 > 15% 7 (70%) 9 (69%) 1.0∆ ABI 0.05 ± 0.23 –0.12 ± 0.30 0.25∆ Pain scale (0–10) –3.7 ± 2.1 –3.6 ± 2.8 0.63∆ QoL (0–100) 18 ± 13 22 ± 17 0.56∆ Rutherford

category (1–6)

–1.28 ± 1.70 –1.36 ± 1.43 0.46

∆ Wound size (cm2) –4.8 ± 3.2 –5.8 ± 5.4 0.59

ABI, ankle–brachial index; BMC, bone marrow cells; QoL, quality-of-life; tcpO

2, transcutaneous oxygen pressure; IA, intra-arterial; IM,

intramuscular; ∆, baseline value at 6 months.

Table 4. Characteristics of Responders Versus Nonresponders

to BMC Delivery

Responders

(n = 27)

Nonresponders

(n = 10) p

BMMC (109) 4.3 ± 1.4 4.1 ± 1.2 0.66

CD34+ (106) 29 ± 15 17 ± 12 0.03

CRP (mg/L) 18 ± 28 100 ± 96 0.03

Leu (109/L) 8.3 ± 2.1 12.3 ± 4.5 0.02

Tr (109/L) 290 ± 107 352 ± 163 0.33

tcpO2 baseline (mmHg) 15 ± 10 9 ± 8 0.07

Wound size (cm2) 8.2 ± 6.8 14.1 ± 10.3 0.12

BMC, bone marrow cells; BMMC, bone marrow mononuclear cells; CRP, C-reactive protein; Leu, leukocytes in peripheral blood; tcpO

2,

transcutaneous oxygen pressure; Tr, thrombocytes in peripheral blood.


corroborate the clinical results of the PROVASA trial

whereby patients with Rutherford classification of 6 CLI

(gangrene or major loss of tissue) at baseline (typical by

the highest inflammatory burden) did not respond to cell

therapy (35). Advanced local inflammation of ischemic tis-

sue acts as a hostile environment for delivered stem cells.

Hence, the question of appropriate timing of stem cell ther-

apy with regard to the deleterious inflammatory setting is

one of the key factors of clinical success.

LIMITATIONS

A relatively small number of patients in individual groups

could be considered to be one of the limitations; the findings

of the present study need to be confirmed in larger, prospec-

tively designed cohorts. Second, absence of a control group

cannot exclude the possibility of spontaneous improvement

in some patients. However, the significant improvements in

tcpO2 as an objective parameter in patients with no options

for surgical or endovascular revascularization are unlikely to

be the result of placebo or spontaneous improvement.

CONCLUSIONS

Both intramuscular and intra-arterial delivery of autolo-

gous BMCs is effective and a comparable therapeutic strategy

for patients with CLI who are not suitable for endovascular

or surgical revascularization. A higher concentration of

CD34+ cells and a lower degree of inflammation are associ-

ated with a better therapeutic response to BMC therapy.

ACKNOWLEDGMENTS: This study was sponsored with a grant from European Regional Development Funding (ITMS code: 26240220023). Authors declare no conflicts of interest.

REFERENCES

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Figure 4. Receiver operating characteristic of CD34+ cells, BMMC count, CRP, and leukocyte levels for the prediction of the BMC therapeutic response. Area under the receiver operating characteristic (ROC) curve: CD34+ = 0.75 (CI 0.57–0.94, p = 0.02); mononuclear cell (MNC) = 0.55 (CI 0.35–0.75, p = 0.65); c-reactive protein (CRP) = 0.26 (CI 0.04–0.47, p = 0.03); leukocyte (leu) = 0.21 (CI 0.01–0.4, p = 0.007)


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