Cytotherapy, 2014; 16: 1733e1738
Comparison of the effect of stem cell therapy and percutaneous transluminal angioplasty on diabetic foot disease in patients with critical limb ischemia
MICHAL DUBSKÝ1, ALEXANDRA JIRKOVSKÁ1, ROBERT BEM1, VLADIMÍRA FEJFAROVÁ1, LIBUSE PAGACOVÁ1, ANDREA NEMCOVÁ1, BEDRICH SIXTA1, JAROSLAV CHLUPAC1, JAN H. PEREGRIN1, EVA SYKOVÁ2 & EDWARD B. JUDE3 1
Institute for Clinical and Experimental Medicine, Prague, Czech Republic, 2Institute of Experimental Medicine, Czech Academy of Science, Prague, Czech Republic, and 3Diabetes Centre, Tameside Hospital NHS Foundation Trust and University of Manchester, Lancashire, United Kingdom
Abstract Background aims. The aim of our study was to compare the effect of autologous stem cell therapy (SCT) and percutaneous transluminal angioplasty (PTA) on diabetic foot disease (DFD) in patients with critical limb ischemia (CLI). Methods. Thirty-one patients with DFD and CLI treated by autologous stem cells and 30 patients treated by PTA were included in the study; 23 patients with the same inclusion criteria who could not undergo PTA or SCT formed the control group. Amputation-free survival, transcutaneous oxygen pressure (TcPO2) and wound healing were assessed over 12 months. Results. Amputation-free survival after 6 and 12 months was significantly greater in the SCT and PTA groups compared with controls (P ¼ 0.001 and P ¼ 0.0029, respectively) without significant differences between the active treatment groups. Increase in TcPO2 did not differ between SCT and PTA groups until 12 months (both Ps < 0.05 compared with baseline), whereas TcPO2 in the control group did not change over the follow-up period. More healed ulcers were observed up to 12 months in the SCT group compared with the PTA and control groups (84 versus 57.7 versus 44.4 %; P ¼ 0.042). Conclusions. Our study showed comparable effects of SCT and PTA on CLI, a major amputation rate that was superior to conservative therapy in patients with diabetic foot and an observable effect of SCT on wound healing. Our results support SCT as a potential promising treatment in patients with CLI and diabetic foot. Key Words: critical limb ischemia, percutaneous transluminal angioplasty, stem cell therapy
Introduction Peripheral arterial disease (PAD) is an important predictor of outcome in patients with diabetes and foot ulceration, and the incidence of severe PAD is estimated to be 50e100/100,000 per year in developed countries [1,2]. The diagnosis and therapy of the most severe form of PAD, critical limb ischemia (CLI), in diabetic patients is difficult because of the absence of pain due to diabetic neuropathy and primarily infrapopliteal lesions [3,4]. Revascularization of severe PAD strongly influenced the rate of limb salvage and was reported in 78% and 85% at 1 year after open and endovascular revascularization, respectively [2,5], compared with a limb-salvage rate at 1 year of 43% in patients with CLI who were not revascularized [6].
The standard treatment methods for CLI include percutaneous transluminal angioplasty (PTA) and vascular bypass, which have been compared in randomized control trials [1,7]; the rate of major amputation and survival did not differ in 3 years of follow-up in two studies. Nevertheless, the therapeutic effect of standard methods of CLI is only partial: almost one-third of patients are not eligible for standard revascularization because of widespread or distally located arterial occlusion or the presence of high-risk co-morbidities [8]. These patients have a poor chance of improvement of CLI when treated conservatively [9], and therefore new therapeutic techniques, such as stem cell therapy (SCT), for these “no-option” patients have been studied in clinical trials.
Correspondence: Michal Dubský, MD, PhD, Institute for Clinical and Experimental Medicine, Videnska 1958/9, 14021 Prague 4, Czech Republic. E-mail: michal.dubsky@gmail.com (Received 28 April 2014; accepted 18 August 2014) http://dx.doi.org/10.1016/j.jcyt.2014.08.010 ISSN 1465-3249 Copyright Ó 2014, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.
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Cell suspension containing vascular precursor cells is the most commonly used cell therapy for CLI. These vascular precursors are characterized by surface markers such as CD34 glycoprotein [10], receptor for vascular endothelial growth factors and surface marker CD133 [11]; other markers are under study [12]. Two methods are currently used to obtain precursor cell suspension, mononuclear fraction of bone marrow-derived mononuclear cells (BMMNC) obtained by trepan biopsy of iliac crest and fraction of mononuclear cells obtained by separation from peripheral blood after previous stimulation by granulocyte-colony stimulating factor (G-CSF, filgrastim, Neupogen, Amgen Inc, Thousand Oaks, CA, USA) for peripheral blood progenitor cells (PBPC). Both types of stem cells have been used for revascularization of ischemic limbs in clinical studies [13e16], and several multicenter randomized controlled trials are ongoing. Recently published meta-analyses have indicated that SCT of ischemic limbs improves limb ischemia, assessed by transcutaneous oxygen pressure (TcPO2) or anklebrachial index, enhances wound healing, decreases both pain and the major amputation rate, and patients with diabetes were included in these findings [3,17,18]. A pilot study of seven patients assessed safety and feasibility of autologous adipose-derived stromal cells in patients with CLI [19]. Our previous experience showed comparable benefits of the two methods of gaining a suspension of autologous stem cells (i.e., harvesting of bone marrow from iliac crest and apheresis of stimulated peripheral blood) in patients with persistent CLI after unsuccessful standard revascularization [20]. We also did not observe an increase in serum levels of pro-angiogenic cytokines during 6 months of followup and no changes in the retina after autologous SCT in terms of induction of systemic angiogenesis [21]. In this study, we focused on comparison of the effect of SCT with the effect of standard revascularization (PTA) in diabetic patients with CLI and treated by SCT who were not eligible for standard revascularization because of angiographic findings. To our knowledge, there are no published data comparing long-term effects of cell therapy with a standard revascularization technique, such as PTA, in diabetic patients with CLI and diabetic foot. The aim of our study was to compare the effects of SCT with PTA and conservative treatment in patients with diabetic foot and persistent ischemia after standard revascularization. Methods Patients attending our foot clinic with diabetic foot ulcers and CLI between January 2008 and December
2012 who received either SCT or PTA were consecutively enrolled into this retrospective study. Patients who fulfilled inclusion criteria and were not eligible for standard revascularization were treated by SCT (n ¼ 31), and patients treated during the study period by PTA (all of whom had previous history of PTA) were included in the re-PTA group (n ¼ 30). The control group (n ¼ 23) comprised patients not eligible for re-PTA who were treated conservatively because of change in the practice of cell treatment in our center, and all of these patients also had a history of PTA. (The change in cell treatment at our center was due to the policy changes of local and European medicine agency recommendations during the inclusion period, which resulted in an interruption of SCT for 11 months.) CLI was defined in accordance with TransAtlantic InterSociety (TASC) II [22] as ulcers or gangrene attributable to objectively proven arterial occlusive disease. All patients had PEDIS (perfusion, extent/ size, depth/tissue loss, infection, and sensation) stage 3 with TcPO2 <30 mm Hg or ankle brachial index <0.6 [23] and Rutherford category 5e6. Patients treated with stem cells gave written informed consent and underwent a protocol of the study that included detailed oncological, hematological and angiographic screening. The study was designed as a comparative retrospective study in patients treated in a single center; it was not randomized because no further vascular intervention was possible in patients treated with SCT. The severity of angiographic findings was assessed in all patients with the Graziani classification [24], and foot ulcers were classified in accordance with the University of Texas Diabetic Wound Classification System [25]. All patients were treated in our complex foot clinic during the follow-up. Those needing pressure off-loading received therapeutic shoes, orthotics or a total contact cast, as indicated. For infected ulcers, we prescribed appropriate antibiotic therapy. Exclusion criteria for SCT were severe limb edema, severe hematologic abnormalities, deep vein thrombosis in the past 6 months and progressive retinopathy or diagnosed neoplasm of any organ. The study was approved by the local ethics committee. SCT was performed by two methods: BMMNC (20 patients) or PBSC obtained by apheresis of peripheral blood stimulated by G-CSF (11 patients). Because we found no difference in outcomes between those methods of isolating stem cells in our previous study [20], data from both groups were pooled for analysis. Bone marrow was taken from the iliac crest by the Jamshidi technique in the operating theater, and
Stem cell therapy versus PTA on diabetic foot disease
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Table I. Baseline patient characteristics.
Parameter
SCT group (n ¼ 31)
PTA group (n ¼ 30)
Control group (n ¼ 23)
P
Age (years) Gender (% men) Diabetes duration (years) HbA1c (mmol/mol) Ischemic heart disease (%) Chronic renal insufficiency—CKD 2e4 (%) Chronic renal failure—dialysis (%) Hypertension (%) Smoking
62.7 10.4 80.6 22.2 14.7 59.9 11.9 61.3 25.8 12.9 83.9 58.1
63.4 11.9 84.3 19.9 7.1 62.5 14.9 59 30 9.7 84.4 56.3
62.7 9.1 77.3 20.3 8.9 60.6 10.6 56.5 34.8 13 82.6 60.8
NS NS NS NS NS NS NS NS NS
CKD, chronic kidney disease, stages 2-4; HbA1c, glycated hemoglobin; NS, not significant.
BMMNCs were separated by using a Smart PReP2 (Harvest Technologies Corporation, Plymouth, MA, USA) or sedimentated using succinated gelatine (Gelofusine, B. Braun, Melsungen, Germany) [26]. PBPCs were separated by leukapheresis on Haemonetics (Braintree, MA, USA) MCSþ after 3e6 days of stimulation by 5e8 mg/kg/daily of G-CSF; minimal concentration of CD34þ cells in peripheral blood before apheresis was 2 104/mL. Final cell suspension of 40e70 mL was injected into the muscles of the affected lower limb in a series of 40e50 punctures of approximately 1e2 mL each and also to the edges of the wound. Effect of treatment of foot ulcers and CLI was assessed by amputation-free survival, changes in TcPO2 and wound healing (number of fully healed patients without major amputation) up to 12 months after enrollment. All data were expressed as mean SD. The statistical significance was analyzed using MannWhitney, Kruskal-Wallis tests and Wilcoxon pair tests with a level of P < 0.05 considered statistically significant.
of co-morbidities (Table I) or in lower limb characteristics except that patients in the SCT and control groups had more severe angiographic findings with higher mean Graziani stage compared with patients in the PTA group (SCT group: 4.8, controls group: 5.0 vs. PTA group: 3.7; both P ¼ 0.02; Table II). Figure 1 summarizes the flow of patients in the study. Of the 31 patients enrolled to SCT group, 3 patients died and 5 underwent major amputation; in the PTA group, 4 patients died, and 5 underwent major amputations. Of the 23 patients in the control group, 2 patients died, and 10 patients underwent major amputation. Amputation-free survival at 6 and 12 months was significantly greater in SCT and PTA groups compared with the control group (P ¼ 0.02, P ¼ 0.0029, respectively; Figure 2), with no difference between the active-treatment groups. TcPO2 in non-amputated patients increased significantly in both treatment groups at 12-month follow-up (from 15.7 to 40.9 mm Hg in the SCT group and from 16.5 to 41.3 mm Hg in the PTA group, both Ps < 0.05), with no significant change in TcPO2 in the control group (from 14.8 to 19.4 mm Hg, P ¼ NS; Figure 3). Severe foot infection with resistant pathogens was the main reason for major amputation in 3 of 5 amputated patients in the SCT group (these revealed a significant increase of TcPO2 even when amputated) and progression of both ischemia and infection was the main reason for
Results There was no difference between groups at baseline in patient characteristics, including age, gender, glycated hemoglobin, duration of diabetes and presence Table II. Baseline limb characteristics.
Parameter TcPO2 at baseline (mm Hg) Rutherford category (mean SD) Angiographic findings—Graziani (mean SD) Area of defect before therapy (cm2) UT Diabetic Wound Classification 2Ce3C (%) 2De3D (%) UT, University of Texas.
SCT group (n ¼ 31) 15.7 5.2 4.8 4.3
10.3 0.2 1.3 1.6
70.9 29.1
PTA group (n ¼ 30) 16.5 5.1 3.7 4.6
71.8 28.2
9 0.1 1.2 1.7
Control group (n ¼ 23)
P
NS NS 0.018 NS
14.8 5.1 5.0 4.1
69.6 30.4
9.4 0.3 0.9 2
NS NS
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Figure 1. Study scheme.
amputation in 2 of 5 patients. There was greater wound healing during follow-up in the SCT group compared with the PTA and control groups at 3 months (46.7 versus 23.3 versus 17.4 %; P ¼ 0.032), 6 months (63.3 versus 34.4 versus 9.5 %; P ¼ 0.005) and 12 months (82.1 versus 57.7 versus. 19.1 %; P ¼ 0.013, P ¼ 0.046, respectively). No serious adverse events were noted after autologous cell therapy, and 1 severe acute bleeding was noted in the PTA group. Other adverse events were observed in 19.3% (n ¼ 6) of patients in the SCT group (transient leg edema, gluteal hematoma after bone marrow harvest) and 13.3% (n ¼ 4) of patients in the PTA group (pseudoaneurysm, dehiscence of groin wound after pseudoaneurysm surgery).
Discussion Our study showed long-term benefit of autologous SCT on limb salvage in CLI that was comparable to
Figure 2. Amputation-free survival among all groups. *P < 0.05, **P < 0.01.
Figure 3. Changes of TcPO2 up to 12 months post-therapy. Black square, SCT group; white triangle, PTA group; black circle, control group. Before, nonsignificant; 6 months, SCT and PTA versus control > P < 0.001; 12 months, P < 0.0001.
repeated PTA. We also observed greater wound healing after SCT compared with PTA despite patients treated by stem cells having significantly worse angiographic findings as assessed by Graziani stages. Both active treatments were superior to conservative therapy. The benefits of SCT on limb salvage has been shown in several meta-analyses. One of the recent meta-analyses assessed 12 randomized controlled trials published through February 2012 [27]. These trials showed reduced amputation rates in the therapeutic arms of those trials, whereas amputation-free survival did not differ. Another randomized clinical trial regarding use of tissue repair cells in PAD patients to treat CLI, RESTORE-CLI [15], showed a significantly longer interval until treatment failure (major amputation, death, new gangrene or worsening of the ulcer) in a group treated with stem cells compared with placebo. The follow-up [28] of the original Therapeutic Angiogenesis by Cell Transplantation study [29] showed higher 3-year survival rates for patients with PAD after BMMNC treatment (80%) compared with typical data presented on the cardiovascular mortality of patients with critical limb ischemia—up to 25% of patients per year. The effect of infrapopliteal PTA on limb salvage was assessed by Peregrin et al. in a large cohort of 1268 patients [30]. Primary and secondary limb salvage rates were 76.1% and 84.4%, respectively; most of the patients had CLI, and 2 or 3 infrapopliteal vessels were occluded. Patients with diabetes and chronic renal failure treated by hemodialysis had significantly lower 1-year limb salvage (66.5%). TcPO2, a standard method of non-invasive measurement of CLI, increased significantly after both stem cell injection and PTA, but no change was observed in the control group. These findings are in
Stem cell therapy versus PTA on diabetic foot disease accordance with published data in which several studies have reported an increase in TcPO2 after cell therapy in patients with CLI [3,17,18]. The PROVASA study [31] of intraarterial progenitor cell transplantation of BMMNC among PAD patients demonstrated significant improvement in TcPO2 and faster wound healing in patients treated by intraarterially injected stem cells compared with placebo, whereas there was no difference in ankle-brachial index values. The mechanism of action of stem cells at the tissue level is not completely clear, but the most probable process is arteriogenesis, an enlargement of pre-existing arterioles induced by increased shear stress due to obstructed main vessels [18,32]. Redlich et al. reported that TcPO2 was a valid predictor for limb salvage in diabetic patients with CLI after infrapopliteal PTA, even when angiographic outcome criteria failed and that ABI measurement was of no prognostic value [33]. Faster wound healing after SCT compared with PTA and conservative therapy could be caused by a paracrine effect of injected precursor cells with local production of angiogenic and proliferative growth factors [34]. All groups had similar ulcers at baseline as defined by the University of Texas Diabetic Wound Classification and similar ulcer size. All plantar ulcers were adequately off-loaded, and clinically infected ulcers were treated with antibiotics. We did not observe any serious adverse events that required admission or caused permanent deterioration of patients’ health after cell treatment; in the PTA group, 1 patient developed acute bleeding at the PTA puncture site. On the basis of the clinical results of many small non-controlled trials, no critical safety concerns appear to exist in relation to the treatment regimens, and no evidence of de-differentiation or tumorigenesis exists related to bone marrowederived cell therapy [3]. Generally, studies about treatment of PAD and diabetic foot disease by stem cells observed few adverse effects. The most common adverse effects described are amputations, which were considered a result of previous disease (diabetic foot and PAD) rather than an adverse effect of SCT [32]. Advantages of our study were well-defined inclusion criteria and methods used for assessment with long-term follow-up. All patients were treated at a single center by standardized methods, and both groups were comparable in demographic characteristics. The limitation of the study was that it was designed as a retrospective comparative study. In conclusion, our study showed a comparable decrease in the rate of major amputation after SCT in no-option CLI patients or after repeated PTA in patients who could be treated with this type of
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revascularization. Both treatment methods showed comparable improvement of CLI during a 1-year follow-up period and were superior to conservative therapy. SCT seems to be more effective in the healing of foot ulcers compared with repeated PTA and non-intervention (control) groups. The question that arises from our study is a consideration of the proper indication for SCT; it may be a promising method in patients with CLI and diabetic foot not only for treatment of ischemia but also for improved wound healing. Larger randomized control trials are needed to confirm the results in this high-risk population. Acknowledgments This study was supported by a grant from the Ministry of Health (MZO 00023001) of the Czech Republic and Foundation of Czech Energy Facilities. Disclosure of interest: The authors have no commercial, proprietary or financial interest in the products or companies described in this article.
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