advance publication journal of atherosclerosis and thrombosis original article ... zx, j...

1Journal of Atherosclerosis and Thrombosis　Vol.21, No. ●

Original Article

Efficacy of Autologous Bone Marrow Mononuclear Cell Therapy in Patients with Peripheral Arterial Disease

Zheng-Xu Wang, Duo Li, Jun-Xia Cao, Yi-Shan Liu, Min Wang, Xiao-Yan Zhang, Jun-Li Li, Hai-Bo Wang, Jin-Long Liu and Bei-Lei Xu

Biotherapy Center, the General Hospital of Beijing Military Command, Beijing, China

Aim: Peripheral arterial disease (PAD), particularly critical limb ischemia (CLI), is a severe cause of amputation and mortality. More than 50% of diabetic patients with CLI die within four to five years. The development of novel stem cell therapies may bring new hope to these patients. We aimed to assess the efficacy of autologous bone marrow cell therapy for treating CLI using a meta-analysis.Methods: We searched the literature in PubMed, the Cochrane Central Registry of Controlled Trials, the Elsevier database and EBSCO for trials of autologous cell therapy in patients with severe PAD published before October 30, 2013. We chose objective clinical endpoints to assess the efficacy of therapy in the meta-analysis, including changes in the ankle-brachial index (ABI), transcutaneous oxygen tension (TcO2), pain scale (0-10 scale) and amputation-free survival (AFS).Results: Thirty-one articles reporting clinical trials involving a total of 1,214 patients treated with bone marrow stem cell-based therapy were collected for the meta-analysis, in which the randomized controlled trials (RCTs) and other trials (non-RCTs) were classified into two groups. Regarding the efficacy of stem cell therapy, the ABI showed significant increases (P＜0.05) at 12 , 24 and 48 weeks after therapy in the non-RCT and RCT groups, but not after four to eight weeks in the non-RCT group. The TcO2 values also increased in the RCT group at four to eight weeks after therapy and 24 weeks after therapy (P＜0.001) and in the non-RCT group at four to eight weeks after therapy (P= 0.01), although no significant increases were observed in the RCT group at 12 weeks after therapy or the non-RCT group at 24 weeks after therapy. Meanwhile, pain was significantly reduced (P＜0.05) at four to eight weeks and 24 weeks after therapy in both the non-RCT and RCT groups, but not at four to eight weeks or 12 weeks after therapy in the RCT group. In addition, the long-term clinical trials demonstrated that the AFS rate improved after therapy with bone marrow stem cells (one-year AFS, P＜0.00001; three-year AFS, P=0.0003). Conclusions: The present results suggest that autologous bone marrow stem cells have an advanta-geous therapy effect in PAD patients who are not eligible for revascularization.

J Atheroscler Thromb, 2014; 21:000-000.

Key words: Autologous bone marrow cell, Stem cell, Meta-analysis, Peripheral arterial disease

Address for correspondence: Zheng-Xu Wang, Biotherapy Center, the General Hospital of Beijing Military Command, No.5 Nan Men Cang Rd., Dongcheng District, Beijing, 100700, China;E-mail: [email protected], [email protected]: December 24, 2013Accepted for publication: May 24, 2014

Introduction

Peripheral arterial disease (PAD) is a common syndrome worldwide, whose incidence is increasing yearly in association with changes in diet and the aging of the population. PAD is consistently associ-ated with a relatively high rate of morbidity and mor-tality due to the effects of symptoms such as claudica-

Advance Publication Journal of Atherosclerosis and Thrombosis Accepted for publication: May 24, 2014 Published online: July 31, 2014

2 Wang et al.

emia.” The search sources included the PubMed data-base, the Cochrane Central Registry of Controlled Trials, the Elsevier database and EBSCO. Papers pub-lished in English and Chinese were included. How-ever, we excluded any case reports and studies using animals or cell lines.

Data Collection and Clinical EndpointsWe performed a meta-analysis of all eligible stud-

ies (randomized controlled trials and other trials, divided into two groups for a separate analysis) and selected objective clinical endpoints to assess the effi-cacy of therapy, such as changes in the ankle-brachial index (ABI), transcutaneous oxygen tension (TcO2), pain scale (0-10 scale) and amputation-free survival (AFS). We collected data regarding safety issues, tak-ing into consideration the incidence of adverse events, including death, recurrence and fever, with different follow-up times.

Statistical AnalysisThe analysis was performed using the Review

Manager Version 5.0 software program (Nordic Cochran Centre, Copenhagen, Denmark). In order to estimate the treatment effects, the outcomes were cal-culated as continuous variables and binary variables with respective 95% confidence intervals (CIs). Tak-ing inherited heterogeneity into consideration, we adopted a random-effects model. The degree of het-erogeneity across the trials was estimated according to the I2 statistics. A P value of ≤ 0.05 was considered to be significant, and the SPSS version 17.0 software program was used to analyze the data according to the chi-square test with likelihood ratios.

Results

Data Selection The literature search yielded 102 references.

Twenty-seven publications were excluded for various reasons (seven were review articles, 14 used animal models, four were case reports and two were meta-analyses). The full text of 75 articles was selected as potentially relevant and retrieved for a more detailed assessment. We excluded 44 studies for not detailing the clinical data or therapy response of the patients. The procedure used to select the clinical trials is shown in Fig.1. Thirty-one articles6, 9-38) reporting clinical trials of bone marrow stem cell-based therapy were ultimately selected for the meta-analysis.

Characteristics of the Patients and Clinical TrialsThe clinical characteristics of the patients

tion, rest pain and gangrene. The major causes of PAD are atherosclerosis obliterans (ASO) and thromboangi-itis obliterans (TAO)1). Critical limb ischemia (CLI) is the most severe stage of PAD, in which plaque forma-tion affects the arteries of critical limbs. Over time, plaque deposits narrow the arteries, thus limiting the flow of oxygen-rich blood, thereby inducing pain and resulting in the development of gangrene with the potential need for amputation. Surgical or endovascu-lar revascularization is the only treatment option in cases of severe disease, although up to 30% of patients are not candidates for such procedures2). The rate of amputation among patients who do not qualify for revascularization is 30-50% at one year2, 3), and approximately 52% of diabetic patients with CLI die during follow-up after four to five years4, 5). In recent years, the development of cell therapy has provided new treatment approaches for such patients. In 2002, Tateishi-Yuyama6) et al. first transplanted autologous bone marrow stem cells in patients with limb ischemia and reported the efficacy of this therapy based on a statistical analysis, providing new hope for patients.

It has also been reported that bone marrow stem cells function via different mechanisms7). For example, transplanted cells migrate to the ischemic endothelial surface, where they improve the density of capillaries and ameliorate the blockage of arteries, primarily by secreting VEGF, which exhibits paracrine effects. In addition, transplanted cells excrete different cytokines in order to regulate the microenvironment, conse-quently alleviating symptoms through their immuno-suppressive role promoted by CD14＋ monocytes. Fur-thermore, bone marrow stem cells acquire myogenic and endothelial properties, with a therapeutic effect based on their capacity for differentiation7). Although the specific mechanisms of new therapies remain uncertain, an increasing number of clinical trials of bone marrow stem cells have demonstrated the critical role of these cells in PAD treatment, and a treatment method based on these principles has recently been entered into a phase Ⅲ clinical trial8). In the present study, we collected and analyzed data and performed a meta-analysis of clinical trials in order to estimate the therapeutic efficacy of bone marrow stem cells in treating PAD, especially CLI.

Methods

Literature SearchThe authors searched for relevant studies of clini-

cal trials published before October 30, 2013 using the search terms “autologous bone marrow stem cells,” “peripheral arterial disease” and “critical limb isch-


3The Therapy of Limb Ischemia by Stem Cells

Table 2. Some of the studies reported detailed data regarding the patients’ disease stage before and after stem cell therapy that could be analyzed with SPSS23, 24), although the majority of the selected studies only described the disease stage before therapy. Using the chi-squared test, we found no significant differences between the treatment groups and control groups in terms of the disease stage (P＞0.05) based on the available data.

ABIThe ankle-brachial index (ABI) is an efficient

tool for objectively documenting the presence of lower extremity PAD. Data for the ABI were available in 20 trials6, 9-14, 17, 18, 21-27, 31, 33-35). We separated the trials into two groups: the RCT group and the non-RCT group. With respect to the efficacy of stem cell therapy com-pared with the control therapy, the estimated pooled MD for the five RCTs6, 21-24) showed a significant increase in the ABI using an inverse variance model (MD 0.14,

recruited for the therapies are summarized in Table 1. Men dominated all of the trials, and the average age of included patients ranged from a minimum of 42 to a maximum of 78 years. Diabetes and hypertension (HT) were the most frequent causes of PAD, followed by smoking; these factors are cited in Table 1. Regard-ing the geographic distribution, 18 trials were per-formed in Asian countries (especially Japan, India and China), eight trials were performed in European coun-tries (Belgium, Italy, Slovakia, Germany, the Czech Republic and Turkey) and five trials were performed in North America (Cuba, USA). In these studies, autologous bone marrow mononuclear and mesenchy-mal stem cells were used for treatment, with a cell dose ranging from 106 to 109. The route of adminis-tration of cells was primarily intramuscular (IM) and intra-arterial (IA). These characteristics are outlined in Table 1.

We also collected data regarding the CLI cate-gory determined according to Fontaine, as shown in

102 studies searched in an electronic database

7 reviews14 animal models4 case reports2 t l2 meta analyseswere excluded

75 clinical trials identified and screened for retrieval

44 trails excluded due to incomplete data

31 trials potentially appropriate for meta analysis (9 controlled trials included)

20 with usable data for ABI12 with usable data for TcO2

Meta-analysis16 with usable data for pain3 with usable data for AFS

Figure 1

Fig.1. Results of the literature search


4 Wang et al.

Table 1. Clinical information of the eligible trials

The table summarizes patient information regarding cases, age, sex and the details of the stem cell therapy, including the type of disease, dose of therapy and injection mode.

Authors and Year AgeNo. of patients

(male)Regimens (per arm)

ComorbiditiesStem cell doses/the route of

administrationH S D

Koji Miyamoto 2006 (Japan)9)

45.8±14.39 8 (7) Bone marrow mononuclear

37.5% 2.0-4.7×109/IM CD34

＋ 6.8±2.6×107

A.S.De Vriese 2008 (Belgium)10)

78±2 16 (8) Bone marrow mononuclear

88% 31% 44% 1.3±0.1×109/IM CD34

＋ 63.2±10.8×106

Serkan Durdu 2006 (Turkey)11) 42.±7.9 28 (25) Bone marrow mononuclear 11% 100% 1.69±0.89×109/IM

Takashi Saigawa 2004 (Japan)12) 62.±10.4 8 (7) Bone marrow mononuclear 100% 75% 3.09-8.15×107/KG/IM

Vishnu Motukuru 2008 (India)13)

38 Bone marrow mononuclear Critical limb ischemia with Buerger’s disease

5.8±4×107/ml/IM CD34

＋ 9.8±9.91×106/40-60ml

G Cobellis 2008 (Italy)14)

60.3±12.4571.89±4.2

10 (6)9 (6)

Bone marrow mononuclear Control

Severe PAD; Ⅲ or Ⅳ Stage of Leriche-Fontaine

109/10ml/IA

Piotr Barc 2006 (Wroctaw)15)

1415

Bone marrow mononuclear Control

Critical limb ischaemia of lower extremities

IM, IA

Alessandro Schiavetta 2012 (Italy)16)

70.5±12.1 60 (44) Bone marrow mononuclear 60% 60% 65% CD34＋ 4.7±3.1×106/IA

Andrej Klepanec 2012 (Slovakia)17)

66±10 41 (35) Bone marrow mononuclear 80% 41% 68% 4.2±1.4×109/IM, IA CD34

＋ 26±14×107

Berthold Amann 2009 (Germany)18)

65±12 51 (34) Bone marrow mononuclear 49% 55% 0.13-6.6×109/IM

Eric Benoit 2011 (USA)19) 69.5 48 (32) Service obtained bone marrow cells Cardiac disease

50% 64.6%

Chenlingzhi 2005 (China)20) 66.9 14 Bone marrow mononuclear 100% IM

Chenbing 2009 (China)21) 64.3±12.7 40 (22) Bone marrow mononuclear 100% 6.1×106−4.5×108/IM

Debin lu 2011 (China)22)

63±865±10

20 (7)21 (8)

Bone marrow mesenchymal stem cellsBone marrow mononuclear

89%95%

61%58%

BMMSC 9.1±1.1×108/IMBMMNC 9.6±1.1×108/IM

Dirk H.Walter 2011 (Germany)23)

64.4±1564.5±16

19 (16)21 (13)

Bone marrow mononuclearPlacebo

74%67%

47%52%

53%48%

IMIM

Emerson C. Perin 2011 (USA)24)

70.92±13.173.86±5.87

11 (6)10 (7)

ALDH bright cellBone marrow mononuclear

45.5%80%

54.5%40%

1.36±0.59×106/IM1.3±1.0×109/IM

Ganyu 2008 (China)25) 71.87±6.35 15 (7) Bone marrow mononuclear Severe chronic lower limb ischemia

Liugeling 2007 (China)26) 72.7 20 (12) Bone marrow mononuclear 100% 1.9×109/50ml/IM

Mark D. Iafrati 2011 (USA)27) 69.5 48 (32) Bone marrow mononuclear 69% 50%

T.Bartsch 2007 (Germany)28)

69±667±11

13 (10)12 (12)

Bone marrow mononuclearControl

100%100%

38.4%41.7%

IM, IA

Tateishi-Yuyama 2002 (Japan)6) 66±12 45 (38) Bone marrow mononuclear 71% 69% 0.7-2.8×109/IM

V.Prochazka 2010 (Czech republic)29)

66.±10.664.1±8.6

42 (36)54 (42)

Bone marrow mononuclearStandard Care

83.3%90.9%

45.2%40.0%

CD34＋ 109/L/IA

Wangguangyu 2009 (China)30) 66.±10.2 83 (49) Bone marrow mononuclear 100% (2.04±0.53)×108/IM

Wangwei 2010 (China)31)

51.8 56 (45) Bone marrow mononuclear 42.9% 0.31-29.46×109/IACD34

＋ 0.09%-1.88%

Wangxiuhui 2011 (China)32)

66.2±9.2 69 (39) Bone marrow mesenchymal stem cells 100% 1.05±0.68×108/per armCD34

＋ 0.67%-1.24%/IM

Xinshijie 2006 (China)33) 65 42 (29) Bone marrow mononuclear 33.3% 1×109−2×109/100ml/IM IM

Xuyifeng 2007 (China)34) 27-86 43 (32) Bone marrow mononuclear 48.8% 79.1% 2.16×107/ml/IM IM



(Cont Table 1)

Authors and Year AgeNo. of patients

(male)Regimens (per arm)

ComorbiditiesStem cell doses/the route of

administrationH S D

Yihai 2011 (China)35) 45 13 (9) Bone marrow mononuclear 38.4% 0.3-1.8×109/per arm/IM IM

Yutaka Saito 2007 (Japan)36) 42.6±8.0 14 (13) Bone marrow mononuclear Ischemic ulcer with Buerger’s disease

8.4±8×107/ml /IM IM

Naomi Idei 2011 (Japan)37)

55.6556.1

51 (39)46 (36)

Bone marrow mononuclearControl

39%48%

78%80%

41%50%

1.8±0.5×109/IMCD34

＋ 3.5±1.4×107 IM

Richard J. Powell 2011 (USA)38)

68.865.9

32 (25)14 (8)

Bone marrow-derived tissue repair cellsPlacebo

84%85.7%

43.8%64.3%

H=hypertension, S= smoke, D=diabetes mellitus

Table 2. Analysis of the results according to the Rutherford CLI category in the available trials

The statistical analyses were performed separately using SPSS.

Study group Fontaine overall Treatment group Control group Chi-square test P (＜0.05)

Alessandro Schiavetta2012

ⅡⅢⅣ

10/6021/6029/60

Eric Benoit2011

ⅡⅢⅣ

18/4830/48

11/3423/34

7/147/14

1.3181.318

0.3300.330

Dirk H.Walter2011

ⅡⅢⅣ

10/4030/40

4/19

15/19

6/21

15/21

0.3010.301

0.7210.721

Mark D.Iarati2011

ⅡⅢⅣ

18/4830/48

Naomi Idei2011

ⅡⅢⅣ

26/9771/97

15/5136/51

11/4635/46

0.3730.373

0.6480.648

Berthold Amann2009

ⅡⅢⅣ

13/5138/51

Xuyifeng 2007

ⅡⅢⅣ

18/4316/439/43

Yutuka Saito2007

ⅡⅢⅣ

2/1412/14

Koji Miyamoto ⅡⅢⅣ

3/118/11

Tateishi Saigama2004

ⅡⅢⅣ

1/83/85/8


6 Wang et al.

stem cells after four to eight, 12 and 24 weeks of fol-low-up (Fig.2A) in the RCT group. In addition, we compared the ABI values obtained at different stages

95% CI 0.07-0.21, P=0.0001; MD 0.14, 95% CI 0.00-0.27, P=0.05; MD 0.14, 95% CI 0.10-0.19, P＜0.00001) among the PAD patients treated with

Figure 2A

Fig.2. Forest plot with 95% CIs of the ABI values (after 4-8 weeks, 12 weeks, 24 weeks and 48 weeks) in the RCT group (A) and the non-RCT group (B)

The random-effects meta-analysis model (Mantel-Haenszel method) was used. MD, mean differ-ence. Each trial is represented by a square, the center of which gives the MD for that trial. The size of the square is proportional to the information in that trial. The ends of the horizontal bars denote the 95% CI. The black diamond gives the overall MD for the combined results of all trials.

Figure 2B

A

B



TcO2

In the RCT group, the transcutaneous oxygen tension (TcO2) values significantly increased after four to eight and 24 weeks (MD 6.89, 95% CI 6.17-7.62, P＜0.00001; MD 20.35, 95% CI 12.51-28.19, P＜0.00001), but not 12 weeks (MD 1.95, 95% CI −7.41-11.30, P=0.68) (Fig.3A). Data regarding the TcO2 values after 12 and 48 weeks were reported in only one study and were not appropriate for a meta-analy-sis16, 29). Meanwhile, the TcO2 values increased in the six selected trials in the non-RCT group12, 13, 16-18, 29) after four to eight weeks of cell therapy (MD 11.90, 95% CI 2.37-21.43, P=0.01), but not 24 weeks of

before and after stem cell treatment in order to assess the effects of therapy in the non-RCT group. The esti-mated pooled MD for the 15 trials9-14, 17, 18, 25-27, 31, 33-35) showed a significant increase in the ABI after 12, 24 and 48 weeks of follow-up (MD 0.12, 95% CI 0.07-0.16, P＜0.00001; MD 0.14, 95% CI 0.10-0.17, P＜0.00001; MD 0.12, 95% CI 0.02-0.23, P=0.02) (Fig.2B). After four to eight weeks, the ABI values were not significantly increased (MD 0.06, 95% CI −0.03-0.16, P=0.21) in the other selected trials in the non-RCT group.

Figure 3A

Fig.3. Forest plot with 95% CIs for the TcO2 values in the RCT group (A) and the non-RCT group (B)

The random-effects meta-analysis model (Mantel-Haenszel method) was used.Figure 3B

A

B


8 Wang et al.

cacy of stem cell therapy. In the patients treated with cell therapy, the level of pain (on a 0-10 scale) signifi-cantly decreased (MD −3.36, 95% CI −5.53-−1.20, P=0.002; MD −3.53, 95% CI −5.65-−1.40, P= 0.001) in the analysis of the 11 studies in the non-RCT group9-11, 13, 17, 20, 25, 26, 29, 31, 35) after four to eight and 24 weeks, although no significant decreases were observed after 12 weeks (MD −1.38, 95% CI −4.00-1.25, P=0.30) (Fig.4B). In terms of the efficacy of

cell therapy (MD 12.82, 95% CI −1.65-27.29, P= 0.08) (Fig.3B).

Pain (Visual Analogue Scale, VAS)The assessment of ischemia in patients with

peripheral arterial disease typically relies on the use of objective surrogate indexes, and the disease burden essentially derives from the level of pain. We collected more subjective symptoms (pain) to examine the effi-

Figure 4A

Fig.4. Forest plot with 95% CIs for pain in the RCT group (A) and the non-RCT group (B) after 4-8 weeks, 12 weeks and 24 weeks

The random-effects meta-analysis model (Mantel-Haenszel method) was used.

Figure 4B

A

B



AFS rate (OR 22.33, 95% CI 4.14-120.5, P=0.0003) (Fig.5).

Safety IssuesWe analyzed the incidence of severe adverse

events in the selected studies. The majority of adverse events were associated with hospitalization for disease process-related complications, not complications related to cell therapy, including pain in the extremities and gastrointestinal disorders that were unrelated to the cell therapy. The adverse events are listed in Table 3 according to the known data, which were obtained from 13 selected studies6, 9, 11, 18, 21, 25, 26, 30-33, 35, 36). As the data show, a total of six of 484 patients died dur-ing follow-up, four patients experienced recurrence and one patient developed a fever after undergoing treatment. The follow-up period ranged from two months to two years, which is expected to have induced variation in the results.

Limitations of our Meta-AnalysisPublication Bias

We cannot eliminate the potential for publica-tion bias, which may have affected the results of the present meta-analysis. Negative trial outcomes are often not published, which may make our efficacy assessment insufficient. In addition, the inclusion of Chinese language studies may have provided another source of publication bias. Furthermore, we selected studies that applied bone marrow stem cell therapies using bone marrow mesenchymal stem cells and bone marrow mononuclear cells, which may contain dis-tinct cell populations.

stem cell therapy compared with the control therapy, the estimated pooled MD for six RCT trials6, 15, 21-24) showed a significant decrease in pain using an inverse variance model (MD −1.37, 95% CI −1.69-−1.04, P＜0.00001) following treatment with stem cells after 24, but not four to eight weeks or 12, weeks of follow-up in the RCT group (Fig.4A) (MD −0.01, 95% CI −1.44-1.43, P=0.99; MD −1.84, 95% CI −4.11-0.44, P=0.11).

Amputation-Free Survival (AFS)The Trans-Atlantic Inter-Society Consensus

(TASC Ⅰ/Ⅱ) suggested that the combined endpoint of amputation-free survival (AFS) is the best outcome measurement for assessing patients with CLI39). Using the data obtained from the long-term clinical trials, we compared the 1-year and 3-year amputation-free survival rates between the stem cell therapy groups and the control groups.

Information regarding the 1-year AFS was avail-able in three trials19, 37, 38) comprising 162 patients (88 of whom received stem cell therapy). The three trials reported higher survival rates for the patients treated with stem cell therapy than those in the control group. The estimated pooled OR for the three trials showed a significant improvement in the one-year amputation-free survival for the patients receiving stem cell ther-apy (OR 8.05, 95% CI 3.58-18.08, P＜0.00001). Meanwhile, information for the 3-year survival rate was available for 97 patients (51 of whom received stem cell therapy). The results of the pooled analysis showed that the patients in the stem cell therapy group exhibited a significantly improved three-year

Fig.5. Forest plot with 95% CIs for the AFS after one and three years in the stem cell therapy and control groups

The random-effects meta-analysis model (Mantel-Haenszel method) was used.Figure 5


10 Wang et al.

cells, has been shown to increase the rate of neo-vascu-larization of ischemic tissue12, 40-43). Several clinical studies have shown that a variety of stem cell types delivered locally via the intramuscular route or sys-temically via the intra-arterial route into ischemic tis-sue have several therapeutic benefits44-46).

We herein presented a comprehensive review and performed a meta-analysis of bone marrow stem cell therapy trials in patients with peripheral arterial dis-ease, particularly CLI. A total of 1,214 patients with-out options for revascularization received treatment in these trials. The analysis, in which the RCTs and other trials (non-RCTs) were classified into two groups, showed that cell therapy significantly improves the ABI, TcO2 and AFS values and reduces rest pain. Regarding the efficacy of stem cell therapy, the ABI values significantly increased (P＜0.05) after 12, 24 and 48 weeks of therapy in both the non-RCT and RCT groups, but not after four to eight weeks of ther-apy in the non-RCT group. In addition, the TcO2 values increased after four to eight and 24 weeks of therapy in the RCT group (P＜0.001) and after four to eight weeks of therapy in the non-RCT group (P=0.01), but not after 12 weeks of therapy in the RCT group or after 24 weeks of therapy in the non-RCT group. Meanwhile, pain was significantly reduced (P＜0.05) after four to eight and 24 weeks of

RobustnessThis meta-analysis was performed using high-

quality studies (randomized controlled studies) and low-quality trials (non-controlled trials). We analyzed the studies separately using the Review Manager Ver-sion 5.0 software program. Given that all of the stud-ies applied different indexes, we selected surrogate objective endpoints (secondary endpoints) included in the trials, such as the ABI, TcO2, AFS and level of pain assessed according to VAS. We excluded end-points without the same criteria.

Discussion

The incidence of CLI is estimated to be almost 500 to 1,000 per million individuals each year. Approximately 50% of patients with CLI undergo amputation within 6-12 months, and approximately 15% of those patients lose the contralateral leg within two years, a condition for which the one-year mortal-ity rate is as high as 20%, rising to 70% and 100% at five and 10 years, respectively15).

Patients who suffer from pain due to CLI are in urgent need of novel therapeutic strategies to preserve their limbs. Transplantation of autologous bone mar-row stem cells, which include both bone marrow mononuclear cells and bone marrow mesenchymal

Table 3. Clinical safety issues in the eligible trials

The table classifies adverse events into three types: death, recurrence and fever, with the follow-up time.

Study group follow-upAdverse event

death Recurrence fever

Tateishi-Yuyama 2002 0/45

Serkan Durdu 2006 0/28

Koji miyamoto 2006 684±549 days 1/8 1/8

Xinshijie 2006 3-24 months 5/42

Yutaka saito 2007 0/14

Liugeling 2007 2-12 months 0/20

Ganyu 2008 6-12 months 0/15 1/15

Wangguangyu 2009 6 months 0/83

Chenbing 2009 0/40

Berthold Amamn 2009 0/51

Wangwei 2010 12 months 0/56 3/56

Yihai 2011 0/13

Wangxiuhui 2011 0/69



cells and injection mode may affect the outcomes of individual trials and thus the heterogeneity of the meta-analysis. All of these factors may have produced bias in this analysis.

Despite the high degree of heterogeneity observed in the current study, this novel therapeutic method may ultimately improve the prognosis of PAD patients, particularly those with CLI. In the present meta-analysis, we confirmed that stem cell therapy is generally effective, resulting in significant changes in each endpoint after treatment.

Conclusion

We herein demonstrated the significant efficacy of autologous bone marrow cell therapy for treating PAD in a meta-analysis, including outcomes showing an increase in ABI and TcO2, a decrease in pain and a longer AFS. These outcomes should be interpreted carefully, as the duration of follow-up was insufficient and the number of available randomized controlled studies was too limited to enable us to confirm the efficacy and safety of this novel strategy for treating PAD. Larger multi-center randomized trials should be conducted in order to verify the effectiveness of this therapy in the near future. The outcomes of our sys-tematic review are expected to encourage further stud-ies of the specific mechanisms by which these cells repair tissues and promote neovascularization, as well as stimulate further research focused on identifying signal pathways associated with the pathological sys-tems of CLI. Such innovations should be expanded and their efficacy verified in multi-center pilot trial studies in order to obtain further evidence of their effectiveness as therapeutic options.

Competing Interests

The authors declare that they have no competing interests.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (No. 31171427 and 30971651 to Zheng-Xu Wang), the Beijing Municipal Science & Technology Project: Clinical Characteristics and Application Research of Capi-tal (No. Z121107001012136 to Zheng-Xu Wang), the National Natural Science Foundation of China (No. 30700974 to Jun-Xia Cao) and the Postdoctoral Foundation of China (No. 20060400775 to Jun-Xia Cao).

therapy in the non-RCT and RCT groups, but not after four to eight weeks or 12 weeks of therapy in the RCT group.

The results of the data analysis showed no major differences between the non-RCT and RCT groups, with an overall trend toward an increase in the ABI and TcO2 values and a decrease in pain. The long-term clinical trials demonstrated improvements in the AFS rate after therapy with bone marrow stem cells (one-year AFS, P＜0.00001; three-year AFS, P=0.0003). Therefore, the meta-analysis showed the potential effi-cacy of autologous bone marrow mononuclear cell therapy in treating peripheral arterial disease.

The inclusion of high-quality randomized con-trolled trials in a meta-analysis allows for more reliable results to be obtained. In the present study, we selected randomized controlled and non-controlled studies due to differences in clinical standards among the studies and the limited number of high-quality trials. Com-pared with a previous meta-analysis1), our meta-analy-sis included only one paper reviewed in that investiga-tion and contained more relevant new studies pub-lished between 2009 and 2013, including recent research carried out in China. In addition, we col-lected long-term follow-up data for the endpoint AFS in order to assess the efficacy of autologous bone mar-row stem cell therapy in patients with peripheral arte-rial disease; this parameter has also been reviewed in three other meta-analyses, all of which showed the same conclusions47-49). Furthermore, we analyzed known safety issues and found that this novel treat-ment is relatively safe, consistent with the results of previous meta-analyses47-49), although the duration of follow-up differed in the selected studies, which makes it difficult to draw the conclusion that stem cell ther-apy is well tolerated. Therefore, larger, placebo-con-trolled, randomized multi-center trials are needed to confirm the safety and efficacy of this method.

The present meta-analysis showed a high rate of heterogeneity. Possible reasons for this phenomenon are as follows. All of the trials were either randomized controlled or non-controlled, and we cannot guaran-tee the quality of data without information for ran-dom and blind allocations. In addition, the patients involved might be in different stages of CLI. As in our review, we included patients diagnosed with Ruther-ford 4 to 6 disease, and the majority of studies pro-vided limited information regarding this issue, which may have led to significant differences between differ-ent groups in certain endpoints, thus affecting the weight of the studies, although we found no differ-ences in regard to the disease stage among the avail-able trials. Moreover, differences in the number of


12 Wang et al.

H, Kodama M, Yoshimura N, Fujiwara H, Namura O, Sogawa M, Hayashi J, Aizawa Y: Clinical application of bone marrow implantation in patients with arteriosclero-sis obliterans, and the association between efficacy and the number of implanted bone marrow cells. Circ J, 2004; 68: 1189-1193

13) Motukuru V, Suresh KR, Vivekanand V, Raj S, Girija KR: Therapeutic angiogenesis in Buerger’s disease (thrombo-angiitis obliterans) patients with critical limb ischemia by autologous transplantation of bone marrow mononuclear cells. J Vasc Surg, 2008; 48: 53S-60S; discussion 60S

14) Cobellis G, Silvestroni A, Lillo S, Sica G, Botti C, Maione C, Schiavone V, Rocco S, Brando G, Sica V: Long-term effects of repeated autologous transplantation of bone marrow cells in patients affected by peripheral arterial dis-ease. Bone Marrow Transplant, 2008; 42: 667-672

15) Barć P, Skóra J, Pupka A, Turkiewicz D, Dorobisz AT, Garcarek J, Tomasiewicz, B, Szyber, P: Bone-marrow cells in therapy of critical limb ischaemia of lower extremities — own experience. Acta Angiol, 2006; 12: 155-166

16) Schiavetta A, Maione C, Botti C, Marino G, Lillo S, Gar-rone A, Lanza L, Pagliari S, Silvestroni A, Signoriello G, Sica V, Cobellis G: A phase Ⅱ trial of autologous trans-plantation of bone marrow stem cells for critical limb ischemia: results of the Naples and Pietra Ligure Evalua-tion of Stem Cells study. Stem Cells Transl Med, 2012; 1: 572-578

17) Klepanec A, Mistrik M, Altaner C, Valachovicova M, Olejarova I, Slysko R, Balazs T, Urlandova T, Hladikova D, Liska B, Tomka J, Vulev I, Madaric J: No difference in intra-arterial and intramuscular delivery of autologous bone marrow cells in patients with advanced critical limb ischemia. Cell Transplant, 2012; 21: 1909-1918

18) Amann B, Luedemann C, Ratei R, Schmidt-Lucke JA: Autologous bone marrow cell transplantation increases leg perfusion and reduces amputations in patients with advanced critical limb ischemia due to peripheral artery disease. Cell Transplant, 2009; 18: 371-380

19) Benoit E, O’Donnell TF Jr, Iafrati MD, Asher E, Bandyk DF, Hallett JW, Lumsden AB, Pearl GJ, Roddy SP, Vijayaraghavan K, Patel AN: The role of amputation as an outcome measure in cellular therapy for critical limb ischemia: implications for clinical trial design. J Transl Med, 2011; 9: 165

20) Chen LZ, Chen CY, Xiao TH: Observation on therapeu-tic effect of autologous bone marrow stem cell transplan-tation for diabetes patients complicated with lower limb ischemia. Chinese Nursing Research, 2005; 19: 1005-1006

21) Chen B, Lu DB, Liang ZW, Jiang YZ, Wang FH, Wu QN, Zhang ZH: Autologous bone marrow mesenchymal stem cell transplantation for treatment of diabetic foot following amplification in vitro. Journal of clinical reha-bilitive tissue engineering research, 2009; 13: 6227-6230

22) Lu D, Chen B, Liang Z, Deng W, Jiang Y, Li S, Xu J, Wu Q, Zhang Z, Xie B, Chen S: Comparison of bone marrow mesenchymal stem cells with bone marrow-derived mononuclear cells for treatment of diabetic critical limb ischemia and foot ulcer: a double-blind, randomized, con-trolled trial. Diabetes Res Clin Pract, 2011; 92: 26-36

23) Walter DH, Krankenberg H, Balzer JO, Kalka C,

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