- Email: [email protected]
Autologous bone marrow mononuclear cell implantation therapy is an effective limb salvage strategy for patients with severe peripheral arterial disease
Autologous bone marrow mononuclear cell implantation therapy is an effective limb salvage strategy for patients with severe peripheral arterial disease Randall W. Franz, MD, FACS, RVT, RPVI,a Kaushal J. Shah, MD, FACS, RPVI,b Richard H. Pin, MD, FACS, RPVI,c Thomas Hankins, CCP,a Jodi F. Hartman, MS,d and Michelle L. Wright, MPH,d Columbus and Westerville, Ohio; Camp Hill, Pa; and Dartmouth, Mass Objective: This study was conducted to determine if intramuscular and intra-arterial stem cell injections delay or prevent major limb amputations, improve ankle-brachial index measurements, relieve rest pain, and improve ulcer healing. Methods: A prospective case series with interventions occurring between December 2007 and September 2012 and a 3-month minimum follow-up was conducted at an urban tertiary care referral hospital. Patients with severe limbthreatening peripheral arterial disease, without other options for revascularization, were eligible for enrollment. Dual intramuscular and intra-arterial injection of bone marrow mononuclear cells harvested from the iliac crest was performed. Major limb amputation at 3 months was the primary outcome measure. Secondary outcome measures included anklebrachial index measurements, rest pain, and ulceration healing. Kaplan-Meier survivorship was performed to ascertain overall survivorship of the procedure. Results: No complications related to the procedure were reported. Of 49 patients (56 limbs) enrolled, two patients (two limbs) died, but had not undergone major amputation, and five limbs (8.9%) underwent major amputation within the first 3 months. Three-month follow-up evaluations were conducted on the remaining 49 limbs (42 patients). Median postprocedure revised Rutherford and Fontaine classifications were significantly lower compared with median baseline classifications. After 3 months, seven patients (nine limbs) died but had not undergone major amputation, and seven limbs (14.3%) underwent major amputation. At a mean follow-up of 18.2 months, the remaining 33 limbs (29 patients) had not undergone a major amputation. Freedom from major adverse limb events (MALE) was 91.1% (95% confidence interval, 79.9-96.2) at 3 months and 75.6% (95% confidence interval, 59.4-86.1) at 12 months. Conclusions: This procedure was designed to improve limb perfusion in an effort to salvage limbs in patients for whom amputation was the only viable treatment option. The results of this analysis indicate that it is an effective strategy for limb salvage for patients with severe peripheral arterial disease. (J Vasc Surg 2015;62:673-80.)
Although revascularization via percutaneous intervention or surgery is the preferred therapeutic option for most patients with critical limb ischemia,1 approximately one-third of these patients are not ideal candidates because of concomitant disease or unfavorable anatomy.2 Eventually, many patients with severe to end-stage peripheral From the OhioHealth Vascular and Vein Center, Columbusa; Vascular Surgery, Geisinger - Holy Spirit Health System, Camp Hillb; Vascular and Endovascular Surgery, Southcoast Hospitals Group, Dartmouthc; and the Orthopaedic Research & Reporting, Ltd, Westerville.d Trial Registration: Use of Autologous Bone-Marrow for Mononuclear Cell Implantation Therapy as a Limb Salvage Procedure in Patients with Moderate to Severe Peripheral Arterial Disease, clinicaltrials.gov Identifier: NCT00919516, http://www.clinicaltrials.gov. Author conflict of interest: none. Correspondence: Randall W. Franz, MD, FACS, RVT, RPVI, OhioHealth Vascular and Vein Center, 285 E State St, Ste 260, Columbus, OH 43215 (e-mail: [email protected]). The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a conflict of interest. 0741-5214 Copyright Ó 2015 by the Society for Vascular Surgery. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.jvs.2015.02.059
arterial occlusive disease (PAD) may resort to limb amputation, which is associated with an array of negative consequences, including significant postoperative morbidity, physical disability, emotional toll, and financial impact.3-5 The median financial cost of managing a patient after amputation is almost twice that of a successful limb salvage.5 Therapeutic angiogenesis using bone marrow mononuclear cells (BM-MNCs) has shown favorable results in patients with critical limb ischemia who are not eligible for other revascularization attempts.6,7 The goal of this therapy is to inject stem cells extracted from BM that are able to differentiate into hematopoietic and mesenchymal cells and to secrete growth factors to promote neoangiogenesis and endothelialization in ischemic tissue.8-12 The conclusion from a metaanalysis of 37 trials involving intramuscular autologous BM-MNC implantation was that the procedure is feasible, relatively safe, and a potentially effective therapeutic strategy for PAD patients for whom traditional revascularization is not possible.13 The authors of another meta-analysis of seven trials (six used intramuscular injection and one used intraarterial injection) suggested that such therapy confers a beneficial effect in patients with severe PAD.14 However, insufficient 673
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evidence was found after a systematic review to support intramuscular injection, mainly due to the small numbers of participants and the lack of trials using the technique in general.15 In December 2007, we began a prospective interventional study to assess a dual intramuscular and intraarterial autologous BM-MNC implantation technique developed by the senior author (R.F.) to treat patients with severe limb-threatening PAD.16 The aim of this procedure was to improve tissue perfusion to salvage limbs in such patients for whom amputation had been recommended. The study objective was to determine if intramuscular and intra-arterial stem cell injections delay or prevent major limb amputations, improve ankle-brachial index (ABI) measurements, relieve rest pain, and improve ulcer healing. Enrollment was completed in September 2012, and the final analysis is presented in this report.
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49 Subjects (56 Limbs) Assessed for Study Eligibility & Enrolled in BM-MNC Study
49 Subjects (56 Limbs) Underwent BM-MNC Implantation
0 Subjects (0 Limbs) Lost to Follow-up 2 Subjects (2 Limbs) Died Before 3-Month Follow-up 5 Subjects (5 Limbs) Underwent Major Amputation before 3-Month Follow-up
42 Subjects (49 Limbs) Included in 3-Month Follow-up Statistical Analysis
Fig 1. Flowchart depicts patient accounting. BM-MNC, Bone marrow mononuclear cell.
METHODS This prospective, interventional case series was designed to assess outcomes of autologous BM-MNC implantation in patients with severe limb-threatening PAD who did not have other options for revascularization. The protocol and informed consent were approved by an Institutional Review Board. A standardized technique was followed for all procedures.16,17 Forty-nine patients (56 limbs) gave written informed consent and were enrolled in the study between December 2007 and September 2012. A flowchart depicting patient accounting is provided in Fig 1. Patients aged $18 years were eligible to participate in the study if the following criteria were met: (1) diagnosis of severe limb-threatening PAD, defined as an ABI measurement of #0.4 or the presence of a nonhealing ischemic ulcer(s), and (2) angiographic identification of stenosis or occlusion of two of the following lower extremity arteries: anterior tibial, posterior tibial, and peroneal. Additional stenosis or occlusion could be present proximally to these vessels. Major limb amputation had been recommended to these patients due to limbthreatening ischemia or unresolved osteomyelitis, ulcerations, or gangrene. Patients with anatomic limitations identified on angiogram that precluded a recommendation of traditional endovascular or open bypass treatments were eligible for inclusion. Excluded from the study were patients aged <18 years and those eligible to undergo traditional endovascular or open bypass for the treatment of PAD, pregnant women, prisoners, patients with developmental challenges, and those unable to consent for participation independently. Eligibility for study participation was determined by each surgeon and approved by the senior author. At enrollment, all patients were undergoing maximum medical therapy for PAD according to the managing vascular surgeon’s discretion and were receiving wound care management, when applicable. While patients were under monitored anesthesia care and local anesthesia, a
three-hole BM needle (DePuy Inc, Warsaw, Ind) with a syringe containing heparin (1000 U/mL) was used to obtain autologous BM aspirate from the anterior superior iliac spine. For patients with heparin-induced thrombocytopenia, only saline was used in the syringe. To concentrate the BM cells, the aspirate (50 to 60 mL) was placed in a centrifuge and spun at 2400 rpm for 12 minutes. The cells were reinjected immediately into the patient, both intravascularly and intramuscularly, using injection sites determined by the location of the stenosis or occlusion, or both, on a prior angiogram. For intramuscular injections, the aspirate (2 mL) was injected intramuscularly into six different lower leg sites in a standardized fashion to approximate the disease surface area. Injections specific to the calf started 8 cm above the medial and lateral malleolus in the midline of the calf and continued proximally in 8-cm increments. For intra-arterial injections, aspirate (12 mL) was injected intravascularly in a 4F catheter in 2-mL increments, flushing between injections. Artery selection was determined by occlusion location: the popliteal artery was used for tibial occlusions, and the common femoral artery was used for superficial femoral artery occlusions. Study design. Patients were evaluated before BM-MNC implantation and then at 2 weeks and 3 months after the procedure. Routine angiography was performed before the procedure to identify the specific location of stenosis or occlusion. At the 2-week postprocedure evaluation, limbs only were assessed for possible complications related to the procedure; no study data were collected. Rest pain and ulcer status, if applicable, were evaluated before the procedure and at 3 months afterward. Revised Rutherford criteria18 were used to classify the extent of acute ischemia, and the Fontaine classification19 was used to categorize PAD. ABIs of the dorsalis pedis artery and posterior tibial artery were measured, when possible.
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Amputations were monitored throughout the study duration. Digit, transmetatarsal, and Chopart amputations were classified as minor amputations, and below-knee and above-knee amputations (BKAs and AKAs) were categorized as major amputations. After the 3-month followup evaluation, monitoring of limb status and subsequent procedures continued for patients not undergoing major limb amputation. The primary outcome measure was major limb amputation at 3 months. Secondary outcome measures included ABI measurement(s), rest pain, and ulceration healing. The intervention was defined as successful if the limb met the following four criteria: improvement of ABI measurement(s), absence of rest pain, absence of ulcers, and absence of major limb amputations. According to a later modification to the study protocol, samples of the autologous BM aspirate were obtained from a subset of patients during BM-MNC implantation to analyze the distribution of leukocytes, erythrocytes, thrombocytes, and monocytes. Statistical analysis. The statistical analysis was performed with Stata 9.2 software (StataCorp LP, College Station, Tex). Descriptive statistics (mean, median, standard deviation [SD], frequency, and percentage) were used to describe demographic and clinical data. Clinical parameters, including revised Rutherford categories, Fontaine classification, and ABI measurements, were matched by patient and compared between preprocedure and postprocedure time intervals. Matched categoric data were compared using the McNemar test. To compare normally distributed continuous variables, a paired t-test was performed, and the Wilcoxon signed rank test was used to compare non-normally distributed variables. Kaplan-Meier survivorship was performed to ascertain overall survivorship of the BM-MNC implantation procedure using two of the Society for Vascular Surgery’s safety objective performance goals for critical limb ischemia treatment20dmajor adverse limb events (MALE) and major above-ankle amputationdas well as postoperative death, as the survival events. Ninety-five percent confidence intervals (CIs) were used throughout the statistical analysis. Statistical differences were considered significant when the P value was #.05 with a power of at least 0.8. RESULTS The study population of 49 patients (56 limbs) consisted of 31 men (63.3%) and 18 women (36.7%), with a mean age of 64.8 years (SD, 13.3; median, 64.6; range, 26.0-92.0 years). Twenty-four patients (49.0%) had a history of using tobacco. The study population had the following distribution of comorbidities: hypertension in 34 (69.4%), diabetes in 29 (59.2%), hyperlipidemia in 22 (44.9%), coronary artery disease in 20 (40.8%), kidney disease/renal failure in 13 (26.5%), cerebrovascular accident in 9 (18.4%), chronic obstructive pulmonary disease in 6 (12.2%), neuropathy in 5 (10.2%), heparin-induced
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thrombocytopenia in 2 (4.1%); and Buerger disease in 2 (4.1%). Twenty-six patients (46.4%) had a history of endovascular procedures, and 23 (41.1%) had a history of major open vascular procedures. At the baseline evaluation, rest pain was observed in 51 limbs (91.1%), was absent in 3 limbs (5.4%) that had ulcers, and could not be assessed in 2 limbs (3.5%) because of paralysis. Fifty-one limbs (91.1%) had 81 nonhealing ulcers, with 29 limbs (51.8%) exhibiting focal gangrene. Location of ulcers was the toe in 54 (66.7%), the foot in 14 (17.3%), and various other lower extremity locations in 13 (16.0%). Forty-six limbs (82.1%) had rest pain and nonhealing ulcers. Baseline revised Rutherford and Fontaine classifications are presented in Tables I and II. The mean baseline revised Rutherford classification was 5.0 (SD, 0.62; median, 5.0; range, 2.0-6.0). The mean baseline Fontaine Classification was 3.9 (SD, 0.41; median, 4.0; range, 2.0-4.0). The mean baseline dorsalis pedis artery ABI measurement was 0.44 (SD, 0.36; median, 0.45; range, 0.00-1.3). The mean baseline posterior tibial artery ABI measurement was 0.39 (SD, 0.37; median, 0.37; range, 0.00-1.8). Baseline ABI measurements could not be obtained in 13 limbs (23.2%)d11 limbs were noncompressible, 1 patient (1 limb) with a wound vacuum refused testing of the posterior tibial artery, and only one of the two arteries was assessed in 2 limbs. Secondary interventions were performed at the time of BMMNC implantation in 13 limbs (23.2%) to increase stem cell delivery to distal vessels. Autologous BM aspirate samples were acquired from 31 limbs (55.4%). The cell count distributions were 93.8 103 mL (SD, 62.3; median, 88.8; range, 25.0258.0) mean thrombocyte volume, 28.5 103 mL (SD, 14.8; median, 27.4; range, 2.5-60.0) mean leukocyte volume, 6.2 103 mL (SD, 2.5; median, 7.1; range, 0.99.0) mean erythrocyte volume, and 1.9 103 mL (SD, 1.1; median, 1.9; range, 0.05-4.1) mean monocyte volume. During the procedure and at the 2-week and 3-month follow-up evaluations, no procedure-related complications were reported. Of the 49 patients (56 limbs) enrolled in the study, 3-month follow-up evaluations could not be conducted for seven patients (seven limbs). Before the 3month follow-up evaluations, two limbs (3.6%) could not be assessed because the patients had died (neither had undergone major amputation) and five limbs (8.9%) underwent major amputations. Three limbs (5.4%) required AKAs at 1.7, 5.4, and 11.0 weeks after the procedure. Two limbs (3.6%) underwent BKAs at 2.6 and 4.1 weeks after the procedure. The remaining 49 limbs (42 patients) underwent the 3-month evaluation. Minor amputations had been performed in 4 (8.1%) of these limbsd2 transmetatarsal (4.1%), 1 Chopart (2.0%), and 1 digit (2.0%). Rest pain remained in 8 limbs (16.3%), was absent in 39 limbs (79.6%), and could not be assessed in 2 limbs (3.5%)
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Table I. Comparison of baseline and 3-month follow-up revised Rutherford classifications of chronic limb ischemia Rutherford classification Grade 0 I II III
Baseline evaluation (n ¼ 56), No. (%)
Category
Clinical description
0 1 2 3 4 5
Asymptomatic Mild claudication Moderate claudication Severe claudication Ischemic rest pain Minor tissue loss, nonhealing ulcer, focal gangrene with diffuse pedal ischemia Major tissue loss, extending above transmetatarsal level, functional foot no longer salvageable
6
0 0 1 0 4 41
3-month follow-up evaluation (n ¼ 48), No. (%)
(0.0) (0.0) (1.8) (0.0) (7.1) (73.2)
8 4 5 12 1 16
10 (17.9)
(16.7) (8.3) (10.4) (25.0) (2.1) (33.3)
2 (4.2)
Table II. Comparison of baseline and 3-month follow-up Fontaine classifications for peripheral arterial disease (PAD) Fontaine classification
Clinical description
I II III IV
Asymptomatic Claudication Rest pain Pedal necrosis
Baseline evaluation (n ¼ 56), No. (%) 0 1 5 50
(0.0) (1.8) (8.9) (89.3)
because of paralysis. Compared with baseline, an 82.2% reduction in patients reporting rest pain was observed at the 3-month follow-up evaluation. Nonhealing ulcers were present in 18 limbs (40.9%) and absent in 26 limbs (59.1%). A 59.1% reduction in the number of patients with nonhealing ulcers was observed at the 3-month follow-up evaluation compared with baseline. The revised Rutherford and Fontaine classifications at the 3-month evaluation are presented in Tables I and II. Although the ulcer healed for one patient with paralysis by the 3-month evaluation, a revised Rutherford classification could not be obtained because rest pain could not be assessed. The mean 3-month revised Rutherford classification was 3.0 (SD, 1.9; median, 3.0; range, 0.0-6.0). The mean 3-month Fontaine classification was 2.6 (SD, 1.2; median, 2.0; range, 1.0-4.0). Statistically significant differences were calculated between the baseline and 3-month follow-up revised Rutherford and Fontaine classifications. The median postprocedure revised Rutherford classification was significantly lower than the median baseline classification (P ¼ .001; power, 1.000). The median postprocedure Fontaine classification was significantly lower than the median baseline classification (P ¼ .001; power, 1.000). The median baseline revised Rutherford and Fontaine classifications corresponded to III5 (minor tissue loss) and IV (pedal necrosis), respectively. At 3 months, the median revised Rutherford and Fontaine classifications improved to I3 (severe claudication) and II (claudication), respectively.
3-month follow-up evaluation (n ¼ 49), No. (%) 9 21 1 18
(18.4) (42.9) (2.0) (36.7)
Three-month follow-up ABI measurements could not be obtained in 16 limbs (32.7%)d10 limbs were noncompressible, ABI measurements were not conducted for 5 limbs of patients who lived out of state, and in 1 limb only one of the two arteries was assessed. The mean 3month ABI measurement was 0.53 (SD, 0.30; median, 0.55; range, 0.00-1.2) for the dorsalis pedis artery and was 0.57 (SD, 0.42; median, 0.55; range, 0.00-1.8) for the posterior tibial artery. Despite these improvements, statistically significant differences were not calculated between median baseline and postprocedure dorsalis pedis (P ¼ .402) and posterior tibial (P ¼ .011; power, 0.514) artery measurements. Using the four-pronged definition of success, defined as improvement of ABI measurement(s), absence of rest pain, absence of ulcers, and absence of major limb amputations, outcomes of the 49 limbs at 3 months after BM-MNC implantation were as follows: 19 limbs (38.8%) met all four criteria, 15 (30.6%) met three criteria, 13 (26.5%) met two criteria; and 2 (4.1%) met one criteria. The limb status of the surviving 49 limbs in 42 patients continued to be monitored. Monitoring subsequently ceased for nine limbs (18.4%) because patients died but had not undergone major amputation. At the time of death, the mean follow-up of this group was 19.2 months (SD, 13.4; median, 13.2; range, 7.4-48.4 months). Among the remaining patients, major amputation was performed in seven limbs (14.3%) at a mean follow-up of 10.8 months (SD, 7.5; median, 7.8; range, 4.1-26.7 months). Three
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Table III. Major amputation distribution for overall study population Outcome No amputations Major amputation <3 months follow-up Between 3 and 6 months follow-up Between 6 and 12 months follow-up >12 months follow-up
No. (%) (N ¼ 56) 44 12 5 1 5 1
(78.6) (21.4) (8.9) (1.4) (8.9) (1.4)
limbs (6.1%) required AKAs and four limbs (8.2%) underwent BKAs. A distribution of the follow-up intervals during which major amputations occurred is presented in Table III. At a mean follow-up of 18.2 months (SD, 12.2; median, 17.0; range, 5.1-52.2 months), 33 limbs in 29 patients had not undergone a major amputation and will continue to be monitored. A minor amputation occurred in three of these limbs (9.1%)done digit (3.0%) and two transmetatarsal (6.1%). Of the 13 limbs (23.2%) for which secondary interventions were performed at the time of BM-MNC implantation, 5 limbs (38.5%) remained intact at the most recent follow-up, whereas 5 limbs (38.5%) required a major amputation, 2 (15.4%) underwent minor amputation, and 1 (7.7%) was associated with a patient who died. Freedom from MALE was 91.1% (95% CI, 79.996.2) at 3 months and 75.6% (95% CI, 59.4-86.1) at 12 months (Fig 2, a). These results also represent freedom of major above-ankle amputation, because no major reinterventions occurred postoperatively. Freedom from postoperative death was 96.1% (95% CI, 85.4-99.0) at 3 months and 83.8% (95% CI, 66.7-92.6) at 12 months (Fig 2, b). The 3- and 12-month freedom from MALE and postoperative death combined was 85.7% (95% CI, 75.6-93.8) and 63.3% (95% CI, 47.2-75.8), respectively (Fig 2, c). DISCUSSION This report presents the final analysis of our study assessing a particular dual intramuscular and intra-arterial autologous BM-MNC implantation technique that began in December 2007. The final results support our study objectives and demonstrate that this form of limb salvage therapy delays or prevents major limb amputations, improves ABI measurements, relieves rest pain, and improves ulcer healing in patients with severe PAD for whom major amputation had been recommended. Of the 56 limbs enrolled, major amputation was avoided in 51 limbs (91.1%) in the first 3 months after the procedure. At a mean most recent follow-up of 18.4 months, major amputation was avoided in 42 limbs (85.7%). Eight of the 49 patients (16.3%) enrolled died during the course of the study, but without undergoing major amputation. Given the complex medical conditions of limb salvage patients, this finding is not unexpected. Although some patients
ultimately required major amputation, it was avoided by a mean of 10.8 months in seven limbs (12.5%). Furthermore, limb perfusion improved in eight limbs (14.3%) such that minor, rather than major, amputation was performed, further extending the survivorship of the lower extremity. ABI measurements improved at the 3-month evaluation, but the difference was not statistically significant. We surmise that the procedure achieves primarily microvascular growth through neoangiogenesis and endothelialization, which results in clinical improvement but does not affect ABI measurements. Compared with baseline, an 82.2% reduction in patients reporting rest pain and a 59.1% reduction in the number of patients with nonhealing ulcers was observed at the 3-month follow-up evaluation. Further evidence of procedure success in improving limb perfusion is shown by the statistically significant improvements in the revised Rutherford and Fontaine classifications. The results of this study are clinically relevant to patients with severe to end-stage PAD for whom major amputation has been recommended. Previously, options for this patient population have been extremely limited. This type of limb salvage therapy involving dual injection of autologous BM-MNC implantation is minimally invasive and, thus, is a reasonable surgical option for patients who often have complex medical histories. Furthermore, it offers hope in increasing limb perfusion to such an extent that a major amputation may be prevented or at least delayed. Extending limb survivorship of this patient population by even a minimum of 3 months improves quality of life. Freedom from MALE was 91.1% (95% CI, 79.9%96.2%) at 3 months and 75.6% (95% CI, 59.4%-86.1%) at 12 months. If difficulty delivering the stem cells was encountered during the procedure, a secondary intervention was performed to improve delivery and to deliver stems cells as distally as possible. It should be noted that this subpopulation of patients experienced a higher incidence of major amputations compared with the overall population, but it is challenging to predetermine when such intraoperative difficulties will be encountered. For this reason and because this is a limb salvage procedure, the patient selection criteria for the procedure has not been modified. Similar BM-MNC implantation studies involving intramuscular injection in patients with severe PAD or chronic limb ischemia have reported limb salvage rates of 53% to 86.3% at 1 year.10,21,22 Although these studies involved patient populations with similar ABIs (range, 0.22-0.33) compared with our study,10,21,22 the study by Higashi et al10 was the only one that reported a similar baseline ulceration rate (>90%). The baseline ulcerations reported by Murphy et al22 and Amann et al22 were much lower, at 31% and 74.5%, respectively. A dual injection technique was chosen to increase cell invasion to the targeted areas intra-arterially and to maximize stem cell delivery locally to ischemic areas.16,17,23,24
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Fig 2. Kaplan-Meier survivorship curve for the bone marrow mononuclear cell (BM-MNC) implantation procedure using freedom from (a) major amputation, (b) postoperative death, and (c) major amputation and postoperative death as survival end points. Because no major vascular reinterventions were performed after BM-MNC implantation, the survival curve for freedom from amputation also represents major above-ankle amputation survivorship.
The intra-arterial injection delivers stem cells to targeted vessels in an antegrade fashion, which directs a high concentration of stem cells to ischemic muscle regions that still are perfused.23 The intramuscular injections at several sites locally disperses a high concentration of stem cells directly into the ischemic musculature. Exclusive use of intra-muscular injections, particularly when confined to the calf, may not affect regions surrounding the femoropopliteal tract or pedal arteries, which often are compromised in patients with advanced, multilevel PAD and may be accessed by intra-arterial injections.24 Conversely, local intramuscular injections are ideal for patients with superficial femoral artery occlusions that may prevent stem cells injected intra-arterially from reaching ischemic muscles.23
The clinical improvements observed in the current study and two previous studies23,24 show this technique of promoting neoangiogenesis through dual physiologic pathways appears effective and should be studied in more detail. Study strengths include complete 3-month follow-up for the study population and continued monitoring of limb status. The main limitation of this study is that a control group was not used. However, because the target patient population for this procedure had end-stage disease and amputation was the only recommended alternative, the senior author did not believe it was ethical to withhold a treatment that offered the possibility of limb salvage. Visual confirmation of neoangiogenesis is difficult given the inherent variability in technique and diagnostic
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imaging protocols. Because of this challenge and also due to the significant comorbidities associated with end-stage PAD patients, angiography was not included as part of the follow-up evaluation. Further study, including endothelial surface marker testing and tissue mitogen assays, is needed to better understand the physiologic response to BM-MNC implantation. Because such testing was beyond the scope of this interventional study, analysis of cell types was added to the study protocol to provide some preliminary data regarding the BM aspirate content. CONCLUSIONS The goal of this dual BM-MNC implantation technique was to improve tissue perfusion to salvage limbs in patients with severe PAD for whom amputation was the only recommended treatment option. The final analysis of our study demonstrates that the procedure effectively delayed or prevented major amputation in the target patient population and, therefore, should be considered as a limb salvage strategy in patients with severe limbthreatening PAD. We acknowledge Alan Parks, DO, Alegent Creighton Health Bergan Mercy Medical Center, Omaha, Nebraska, for contributing to the concept and design of the study and data acquisition, and Jason D. Johnson, DO, Fellow, Plastic and Reconstructive Surgery, Philadelphia College of Osteopathic Medicine, Philadelphia, Pennsylvania, for contributing to data acquisition. AUTHOR CONTRIBUTIONS Conception and design: RF, KS, TH Analysis and interpretation: RF, KS, RP, TH, JH, MW Data collection: RF, TH, JH, MW Writing the article: RF, JH, MW Critical revision of the article: RF, KS, RP, TH, JH, MW Final approval of the article: RF, KS, RP, TH, JH, MW Statistical analysis: MW Obtained funding: Not applicable Overall responsibility: RF REFERENCES 1. Attanasio S, Snell J. Therapeutic angiogenesis in the management of critical limb ischemia: current concepts and review. Cardiol Rev 2009;17:115-20. 2. Fadini GP, Agostini C, Sartore S, Avogaro A. Endothelial progenitor cells in the natural history of atherosclerosis. Atherosclerosis 2007;194: 46-54. 3. De Vries M, Ouwendijk R, Kessels AG, de Haan MW, Flobbe K, Hunink MG, et al. Comparison of generic and disease-specific questionnaires for the assessment of quality of life in patients with peripheral arterial disease. J Vasc Surg 2005;41:261-8. 4. Feinglass J, Pearce WH, Martin GJ, Gibbs J, Cowper D, Sorensen M, et al. Postoperative and late survival outcomes after major amputation: findings from the Department of Veterans Affairs National Surgical Quality Improvement Program. Surgery 2001;130:21-9.
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5. Singh S, Evans L, Datta D, Gaines P, Beard JD. The costs of managing lower limb-threatening ischaemia. Eur J Vasc Endovasc Surg 1996;12: 359-62. 6. Kajiguchi M, Kondo T, Izawa H, Kobayashi M, Yamamoto K, Shintani S, et al. Safety and efficacy of autologous progenitor cell transplantation for therapeutic angiogenesis in patients with critical limb ischemia. Circ J 2007;71:196-201. 7. Napoli C, Farzati B, Sica V, Iannuzzi E, Coppola G, Silvestroni A, et al. Beneficial effects of autologous bone marrow cell infusion and antioxidants/L-arginine in patients with chronic critical limb ischemia. Eur J Cardiovasc Prev Rehabil 2008;15:709-18. 8. Esato K, Hamano K, Li TS, Furutani A, Seyama A, Takenaka H, et al. Neovascularization induced by autologous bone marrow cell implantation in peripheral arterial disease. Cell Transplant 2002;11: 747-52. 9. Hamano K, Li TS, Kobayashi T, Tanaka N, Kobayashi S, Matsuzaki M, et al. The induction of angiogenesis by the implantation of autologous bone marrow cells: a novel and simple therapeutic method. Surgery 2001;130:44-54. 10. Higashi Y, Kimura M, Hara K, Noma K, Jitsuiki D, Nakagawa K, et al. Autologous bone-marrow mononuclear cell implantation improves endothelium-dependent vasodilation in patients with limb ischemia. Circulation 2004;109:1215-8. 11. Tateishi-Yuyama E, Matsubara H, Murohara T, Ikeda U, Shintani S, Masaki H, et al. Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial. Lancet 2002;360: 427-35. 12. Iwase T, Nagaya N, Fujii T, Itoh T, Ishibashi-Ueda H, Yamagishi M, et al. Adrenomedullin enhances angiogenic potency of bone marrow transplantation in a rat model of hindlimb ischemia. Circulation 2005;111:356-62. 13. Fadini GP, Agostini C, Avogaro A. Autologous stem cell therapy for peripheral arterial disease meta-analysis and systematic review of the literature. Atherosclerosis 2010;209:10-7. 14. Wen Y, Meng L, Gao Q. Autologous bone marrow cell therapy for patients with peripheral arterial disease: a meta-analysis of randomized controlled trials. Expert Opin Biol Ther 2011;11:1581-9. 15. Moazzami K, Majdzadeh R, Nedjat S. Local intramuscular transplantation of autologous mononuclear cells for critical lower limb ischaemia. Cochrane Database Syst Rev 2011:CD008347. 16. Franz RW, Parks A, Shah KJ, Hankins T, Hartman JF, Wright ML. Use of autologous bone marrow mononuclear cell implantation therapy as a limb salvage procedure in patients with severe peripheral arterial disease. J Vasc Surg 2009;50:1378-90. 17. Franz RW, Shah KJ, Johnson JD, Pin RH, Parks AM, Hankins T, et al. Short- to mid-term results using autologous bone-marrow mononuclear cell implantation therapy as a limb salvage procedure in patients with severe peripheral arterial disease. Vasc Endovascular Surg 2011;45:398-406. 18. Rutherford RB, Baker JD, Ernst C, Johnston KW, Porter JM, Ahn S, et al. Recommended standards for reports dealing with lower extremity ischemia: revised version. J Vasc Surg 1997;26:517-38. 19. Rutherford RB, Flanigan DP, Gupta SK, Johnston KW, Karmody A, Whittemore AD, et al. Suggested standards for reports dealing with lower extremity ischemia. J Vasc Surg 1986;4:80-94. 20. Goodney PP, Schanzer A, Demartino RR, Nolan BW, Hevelone ND, Conte MS, et al; Vascular Study Group of New England. Validation of the Society for Vascular Surgery’s objective performance goals for critical limb ischemia in everyday vascular surgery practice. J Vasc Surg 2011;54:100-8. 21. 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;18:371-80. 22. Murphy MP, Lawson JH, Rapp BM, Dalsing MC, Klein J, Wilson MG, et al. Autologous bone marrow mononuclear cell therapy is safe and promotes amputation-free survival in patients with critical limb ischemia. J Vasc Surg 2011;53:1565-74.
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23. Bartsch T, Brehm M, Zeus T, Kogler G, Wernet P, Strauer BE. Transplantation of autologous mononuclear bone marrow stem cells in patients with peripheral arterial disease (The TAM-PAD study). Clin Res Cardiol 2007;96:891-9. 24. Van Tongeren RB, Hamming JF, Fibbe WE, Van Weel V, Frerichs SJ, Stiggelbout AM, et al. Intramuscular or combined intramuscular/intra-
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arterial administration of bone marrow mononuclear cells: a clinical trial in patients with advanced limb ischemia. J Cardiovasc Surg 2008;49: 51-8.
Submitted Dec 15, 2014; accepted Feb 13, 2015.
Although revascularization via percutaneous intervention or surgery is the preferred therapeutic option for most patients with critical limb ischemia,1 approximately one-third of these patients are not ideal candidates because of concomitant disease or unfavorable anatomy.2 Eventually, many patients with severe to end-stage peripheral From the OhioHealth Vascular and Vein Center, Columbusa; Vascular Surgery, Geisinger - Holy Spirit Health System, Camp Hillb; Vascular and Endovascular Surgery, Southcoast Hospitals Group, Dartmouthc; and the Orthopaedic Research & Reporting, Ltd, Westerville.d Trial Registration: Use of Autologous Bone-Marrow for Mononuclear Cell Implantation Therapy as a Limb Salvage Procedure in Patients with Moderate to Severe Peripheral Arterial Disease, clinicaltrials.gov Identifier: NCT00919516, http://www.clinicaltrials.gov. Author conflict of interest: none. Correspondence: Randall W. Franz, MD, FACS, RVT, RPVI, OhioHealth Vascular and Vein Center, 285 E State St, Ste 260, Columbus, OH 43215 (e-mail: [email protected]). The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a conflict of interest. 0741-5214 Copyright Ó 2015 by the Society for Vascular Surgery. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.jvs.2015.02.059
arterial occlusive disease (PAD) may resort to limb amputation, which is associated with an array of negative consequences, including significant postoperative morbidity, physical disability, emotional toll, and financial impact.3-5 The median financial cost of managing a patient after amputation is almost twice that of a successful limb salvage.5 Therapeutic angiogenesis using bone marrow mononuclear cells (BM-MNCs) has shown favorable results in patients with critical limb ischemia who are not eligible for other revascularization attempts.6,7 The goal of this therapy is to inject stem cells extracted from BM that are able to differentiate into hematopoietic and mesenchymal cells and to secrete growth factors to promote neoangiogenesis and endothelialization in ischemic tissue.8-12 The conclusion from a metaanalysis of 37 trials involving intramuscular autologous BM-MNC implantation was that the procedure is feasible, relatively safe, and a potentially effective therapeutic strategy for PAD patients for whom traditional revascularization is not possible.13 The authors of another meta-analysis of seven trials (six used intramuscular injection and one used intraarterial injection) suggested that such therapy confers a beneficial effect in patients with severe PAD.14 However, insufficient 673
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evidence was found after a systematic review to support intramuscular injection, mainly due to the small numbers of participants and the lack of trials using the technique in general.15 In December 2007, we began a prospective interventional study to assess a dual intramuscular and intraarterial autologous BM-MNC implantation technique developed by the senior author (R.F.) to treat patients with severe limb-threatening PAD.16 The aim of this procedure was to improve tissue perfusion to salvage limbs in such patients for whom amputation had been recommended. The study objective was to determine if intramuscular and intra-arterial stem cell injections delay or prevent major limb amputations, improve ankle-brachial index (ABI) measurements, relieve rest pain, and improve ulcer healing. Enrollment was completed in September 2012, and the final analysis is presented in this report.
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49 Subjects (56 Limbs) Assessed for Study Eligibility & Enrolled in BM-MNC Study
49 Subjects (56 Limbs) Underwent BM-MNC Implantation
0 Subjects (0 Limbs) Lost to Follow-up 2 Subjects (2 Limbs) Died Before 3-Month Follow-up 5 Subjects (5 Limbs) Underwent Major Amputation before 3-Month Follow-up
42 Subjects (49 Limbs) Included in 3-Month Follow-up Statistical Analysis
Fig 1. Flowchart depicts patient accounting. BM-MNC, Bone marrow mononuclear cell.
METHODS This prospective, interventional case series was designed to assess outcomes of autologous BM-MNC implantation in patients with severe limb-threatening PAD who did not have other options for revascularization. The protocol and informed consent were approved by an Institutional Review Board. A standardized technique was followed for all procedures.16,17 Forty-nine patients (56 limbs) gave written informed consent and were enrolled in the study between December 2007 and September 2012. A flowchart depicting patient accounting is provided in Fig 1. Patients aged $18 years were eligible to participate in the study if the following criteria were met: (1) diagnosis of severe limb-threatening PAD, defined as an ABI measurement of #0.4 or the presence of a nonhealing ischemic ulcer(s), and (2) angiographic identification of stenosis or occlusion of two of the following lower extremity arteries: anterior tibial, posterior tibial, and peroneal. Additional stenosis or occlusion could be present proximally to these vessels. Major limb amputation had been recommended to these patients due to limbthreatening ischemia or unresolved osteomyelitis, ulcerations, or gangrene. Patients with anatomic limitations identified on angiogram that precluded a recommendation of traditional endovascular or open bypass treatments were eligible for inclusion. Excluded from the study were patients aged <18 years and those eligible to undergo traditional endovascular or open bypass for the treatment of PAD, pregnant women, prisoners, patients with developmental challenges, and those unable to consent for participation independently. Eligibility for study participation was determined by each surgeon and approved by the senior author. At enrollment, all patients were undergoing maximum medical therapy for PAD according to the managing vascular surgeon’s discretion and were receiving wound care management, when applicable. While patients were under monitored anesthesia care and local anesthesia, a
three-hole BM needle (DePuy Inc, Warsaw, Ind) with a syringe containing heparin (1000 U/mL) was used to obtain autologous BM aspirate from the anterior superior iliac spine. For patients with heparin-induced thrombocytopenia, only saline was used in the syringe. To concentrate the BM cells, the aspirate (50 to 60 mL) was placed in a centrifuge and spun at 2400 rpm for 12 minutes. The cells were reinjected immediately into the patient, both intravascularly and intramuscularly, using injection sites determined by the location of the stenosis or occlusion, or both, on a prior angiogram. For intramuscular injections, the aspirate (2 mL) was injected intramuscularly into six different lower leg sites in a standardized fashion to approximate the disease surface area. Injections specific to the calf started 8 cm above the medial and lateral malleolus in the midline of the calf and continued proximally in 8-cm increments. For intra-arterial injections, aspirate (12 mL) was injected intravascularly in a 4F catheter in 2-mL increments, flushing between injections. Artery selection was determined by occlusion location: the popliteal artery was used for tibial occlusions, and the common femoral artery was used for superficial femoral artery occlusions. Study design. Patients were evaluated before BM-MNC implantation and then at 2 weeks and 3 months after the procedure. Routine angiography was performed before the procedure to identify the specific location of stenosis or occlusion. At the 2-week postprocedure evaluation, limbs only were assessed for possible complications related to the procedure; no study data were collected. Rest pain and ulcer status, if applicable, were evaluated before the procedure and at 3 months afterward. Revised Rutherford criteria18 were used to classify the extent of acute ischemia, and the Fontaine classification19 was used to categorize PAD. ABIs of the dorsalis pedis artery and posterior tibial artery were measured, when possible.
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Amputations were monitored throughout the study duration. Digit, transmetatarsal, and Chopart amputations were classified as minor amputations, and below-knee and above-knee amputations (BKAs and AKAs) were categorized as major amputations. After the 3-month followup evaluation, monitoring of limb status and subsequent procedures continued for patients not undergoing major limb amputation. The primary outcome measure was major limb amputation at 3 months. Secondary outcome measures included ABI measurement(s), rest pain, and ulceration healing. The intervention was defined as successful if the limb met the following four criteria: improvement of ABI measurement(s), absence of rest pain, absence of ulcers, and absence of major limb amputations. According to a later modification to the study protocol, samples of the autologous BM aspirate were obtained from a subset of patients during BM-MNC implantation to analyze the distribution of leukocytes, erythrocytes, thrombocytes, and monocytes. Statistical analysis. The statistical analysis was performed with Stata 9.2 software (StataCorp LP, College Station, Tex). Descriptive statistics (mean, median, standard deviation [SD], frequency, and percentage) were used to describe demographic and clinical data. Clinical parameters, including revised Rutherford categories, Fontaine classification, and ABI measurements, were matched by patient and compared between preprocedure and postprocedure time intervals. Matched categoric data were compared using the McNemar test. To compare normally distributed continuous variables, a paired t-test was performed, and the Wilcoxon signed rank test was used to compare non-normally distributed variables. Kaplan-Meier survivorship was performed to ascertain overall survivorship of the BM-MNC implantation procedure using two of the Society for Vascular Surgery’s safety objective performance goals for critical limb ischemia treatment20dmajor adverse limb events (MALE) and major above-ankle amputationdas well as postoperative death, as the survival events. Ninety-five percent confidence intervals (CIs) were used throughout the statistical analysis. Statistical differences were considered significant when the P value was #.05 with a power of at least 0.8. RESULTS The study population of 49 patients (56 limbs) consisted of 31 men (63.3%) and 18 women (36.7%), with a mean age of 64.8 years (SD, 13.3; median, 64.6; range, 26.0-92.0 years). Twenty-four patients (49.0%) had a history of using tobacco. The study population had the following distribution of comorbidities: hypertension in 34 (69.4%), diabetes in 29 (59.2%), hyperlipidemia in 22 (44.9%), coronary artery disease in 20 (40.8%), kidney disease/renal failure in 13 (26.5%), cerebrovascular accident in 9 (18.4%), chronic obstructive pulmonary disease in 6 (12.2%), neuropathy in 5 (10.2%), heparin-induced
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thrombocytopenia in 2 (4.1%); and Buerger disease in 2 (4.1%). Twenty-six patients (46.4%) had a history of endovascular procedures, and 23 (41.1%) had a history of major open vascular procedures. At the baseline evaluation, rest pain was observed in 51 limbs (91.1%), was absent in 3 limbs (5.4%) that had ulcers, and could not be assessed in 2 limbs (3.5%) because of paralysis. Fifty-one limbs (91.1%) had 81 nonhealing ulcers, with 29 limbs (51.8%) exhibiting focal gangrene. Location of ulcers was the toe in 54 (66.7%), the foot in 14 (17.3%), and various other lower extremity locations in 13 (16.0%). Forty-six limbs (82.1%) had rest pain and nonhealing ulcers. Baseline revised Rutherford and Fontaine classifications are presented in Tables I and II. The mean baseline revised Rutherford classification was 5.0 (SD, 0.62; median, 5.0; range, 2.0-6.0). The mean baseline Fontaine Classification was 3.9 (SD, 0.41; median, 4.0; range, 2.0-4.0). The mean baseline dorsalis pedis artery ABI measurement was 0.44 (SD, 0.36; median, 0.45; range, 0.00-1.3). The mean baseline posterior tibial artery ABI measurement was 0.39 (SD, 0.37; median, 0.37; range, 0.00-1.8). Baseline ABI measurements could not be obtained in 13 limbs (23.2%)d11 limbs were noncompressible, 1 patient (1 limb) with a wound vacuum refused testing of the posterior tibial artery, and only one of the two arteries was assessed in 2 limbs. Secondary interventions were performed at the time of BMMNC implantation in 13 limbs (23.2%) to increase stem cell delivery to distal vessels. Autologous BM aspirate samples were acquired from 31 limbs (55.4%). The cell count distributions were 93.8 103 mL (SD, 62.3; median, 88.8; range, 25.0258.0) mean thrombocyte volume, 28.5 103 mL (SD, 14.8; median, 27.4; range, 2.5-60.0) mean leukocyte volume, 6.2 103 mL (SD, 2.5; median, 7.1; range, 0.99.0) mean erythrocyte volume, and 1.9 103 mL (SD, 1.1; median, 1.9; range, 0.05-4.1) mean monocyte volume. During the procedure and at the 2-week and 3-month follow-up evaluations, no procedure-related complications were reported. Of the 49 patients (56 limbs) enrolled in the study, 3-month follow-up evaluations could not be conducted for seven patients (seven limbs). Before the 3month follow-up evaluations, two limbs (3.6%) could not be assessed because the patients had died (neither had undergone major amputation) and five limbs (8.9%) underwent major amputations. Three limbs (5.4%) required AKAs at 1.7, 5.4, and 11.0 weeks after the procedure. Two limbs (3.6%) underwent BKAs at 2.6 and 4.1 weeks after the procedure. The remaining 49 limbs (42 patients) underwent the 3-month evaluation. Minor amputations had been performed in 4 (8.1%) of these limbsd2 transmetatarsal (4.1%), 1 Chopart (2.0%), and 1 digit (2.0%). Rest pain remained in 8 limbs (16.3%), was absent in 39 limbs (79.6%), and could not be assessed in 2 limbs (3.5%)
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Table I. Comparison of baseline and 3-month follow-up revised Rutherford classifications of chronic limb ischemia Rutherford classification Grade 0 I II III
Baseline evaluation (n ¼ 56), No. (%)
Category
Clinical description
0 1 2 3 4 5
Asymptomatic Mild claudication Moderate claudication Severe claudication Ischemic rest pain Minor tissue loss, nonhealing ulcer, focal gangrene with diffuse pedal ischemia Major tissue loss, extending above transmetatarsal level, functional foot no longer salvageable
6
0 0 1 0 4 41
3-month follow-up evaluation (n ¼ 48), No. (%)
(0.0) (0.0) (1.8) (0.0) (7.1) (73.2)
8 4 5 12 1 16
10 (17.9)
(16.7) (8.3) (10.4) (25.0) (2.1) (33.3)
2 (4.2)
Table II. Comparison of baseline and 3-month follow-up Fontaine classifications for peripheral arterial disease (PAD) Fontaine classification
Clinical description
I II III IV
Asymptomatic Claudication Rest pain Pedal necrosis
Baseline evaluation (n ¼ 56), No. (%) 0 1 5 50
(0.0) (1.8) (8.9) (89.3)
because of paralysis. Compared with baseline, an 82.2% reduction in patients reporting rest pain was observed at the 3-month follow-up evaluation. Nonhealing ulcers were present in 18 limbs (40.9%) and absent in 26 limbs (59.1%). A 59.1% reduction in the number of patients with nonhealing ulcers was observed at the 3-month follow-up evaluation compared with baseline. The revised Rutherford and Fontaine classifications at the 3-month evaluation are presented in Tables I and II. Although the ulcer healed for one patient with paralysis by the 3-month evaluation, a revised Rutherford classification could not be obtained because rest pain could not be assessed. The mean 3-month revised Rutherford classification was 3.0 (SD, 1.9; median, 3.0; range, 0.0-6.0). The mean 3-month Fontaine classification was 2.6 (SD, 1.2; median, 2.0; range, 1.0-4.0). Statistically significant differences were calculated between the baseline and 3-month follow-up revised Rutherford and Fontaine classifications. The median postprocedure revised Rutherford classification was significantly lower than the median baseline classification (P ¼ .001; power, 1.000). The median postprocedure Fontaine classification was significantly lower than the median baseline classification (P ¼ .001; power, 1.000). The median baseline revised Rutherford and Fontaine classifications corresponded to III5 (minor tissue loss) and IV (pedal necrosis), respectively. At 3 months, the median revised Rutherford and Fontaine classifications improved to I3 (severe claudication) and II (claudication), respectively.
3-month follow-up evaluation (n ¼ 49), No. (%) 9 21 1 18
(18.4) (42.9) (2.0) (36.7)
Three-month follow-up ABI measurements could not be obtained in 16 limbs (32.7%)d10 limbs were noncompressible, ABI measurements were not conducted for 5 limbs of patients who lived out of state, and in 1 limb only one of the two arteries was assessed. The mean 3month ABI measurement was 0.53 (SD, 0.30; median, 0.55; range, 0.00-1.2) for the dorsalis pedis artery and was 0.57 (SD, 0.42; median, 0.55; range, 0.00-1.8) for the posterior tibial artery. Despite these improvements, statistically significant differences were not calculated between median baseline and postprocedure dorsalis pedis (P ¼ .402) and posterior tibial (P ¼ .011; power, 0.514) artery measurements. Using the four-pronged definition of success, defined as improvement of ABI measurement(s), absence of rest pain, absence of ulcers, and absence of major limb amputations, outcomes of the 49 limbs at 3 months after BM-MNC implantation were as follows: 19 limbs (38.8%) met all four criteria, 15 (30.6%) met three criteria, 13 (26.5%) met two criteria; and 2 (4.1%) met one criteria. The limb status of the surviving 49 limbs in 42 patients continued to be monitored. Monitoring subsequently ceased for nine limbs (18.4%) because patients died but had not undergone major amputation. At the time of death, the mean follow-up of this group was 19.2 months (SD, 13.4; median, 13.2; range, 7.4-48.4 months). Among the remaining patients, major amputation was performed in seven limbs (14.3%) at a mean follow-up of 10.8 months (SD, 7.5; median, 7.8; range, 4.1-26.7 months). Three
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Table III. Major amputation distribution for overall study population Outcome No amputations Major amputation <3 months follow-up Between 3 and 6 months follow-up Between 6 and 12 months follow-up >12 months follow-up
No. (%) (N ¼ 56) 44 12 5 1 5 1
(78.6) (21.4) (8.9) (1.4) (8.9) (1.4)
limbs (6.1%) required AKAs and four limbs (8.2%) underwent BKAs. A distribution of the follow-up intervals during which major amputations occurred is presented in Table III. At a mean follow-up of 18.2 months (SD, 12.2; median, 17.0; range, 5.1-52.2 months), 33 limbs in 29 patients had not undergone a major amputation and will continue to be monitored. A minor amputation occurred in three of these limbs (9.1%)done digit (3.0%) and two transmetatarsal (6.1%). Of the 13 limbs (23.2%) for which secondary interventions were performed at the time of BM-MNC implantation, 5 limbs (38.5%) remained intact at the most recent follow-up, whereas 5 limbs (38.5%) required a major amputation, 2 (15.4%) underwent minor amputation, and 1 (7.7%) was associated with a patient who died. Freedom from MALE was 91.1% (95% CI, 79.996.2) at 3 months and 75.6% (95% CI, 59.4-86.1) at 12 months (Fig 2, a). These results also represent freedom of major above-ankle amputation, because no major reinterventions occurred postoperatively. Freedom from postoperative death was 96.1% (95% CI, 85.4-99.0) at 3 months and 83.8% (95% CI, 66.7-92.6) at 12 months (Fig 2, b). The 3- and 12-month freedom from MALE and postoperative death combined was 85.7% (95% CI, 75.6-93.8) and 63.3% (95% CI, 47.2-75.8), respectively (Fig 2, c). DISCUSSION This report presents the final analysis of our study assessing a particular dual intramuscular and intra-arterial autologous BM-MNC implantation technique that began in December 2007. The final results support our study objectives and demonstrate that this form of limb salvage therapy delays or prevents major limb amputations, improves ABI measurements, relieves rest pain, and improves ulcer healing in patients with severe PAD for whom major amputation had been recommended. Of the 56 limbs enrolled, major amputation was avoided in 51 limbs (91.1%) in the first 3 months after the procedure. At a mean most recent follow-up of 18.4 months, major amputation was avoided in 42 limbs (85.7%). Eight of the 49 patients (16.3%) enrolled died during the course of the study, but without undergoing major amputation. Given the complex medical conditions of limb salvage patients, this finding is not unexpected. Although some patients
ultimately required major amputation, it was avoided by a mean of 10.8 months in seven limbs (12.5%). Furthermore, limb perfusion improved in eight limbs (14.3%) such that minor, rather than major, amputation was performed, further extending the survivorship of the lower extremity. ABI measurements improved at the 3-month evaluation, but the difference was not statistically significant. We surmise that the procedure achieves primarily microvascular growth through neoangiogenesis and endothelialization, which results in clinical improvement but does not affect ABI measurements. Compared with baseline, an 82.2% reduction in patients reporting rest pain and a 59.1% reduction in the number of patients with nonhealing ulcers was observed at the 3-month follow-up evaluation. Further evidence of procedure success in improving limb perfusion is shown by the statistically significant improvements in the revised Rutherford and Fontaine classifications. The results of this study are clinically relevant to patients with severe to end-stage PAD for whom major amputation has been recommended. Previously, options for this patient population have been extremely limited. This type of limb salvage therapy involving dual injection of autologous BM-MNC implantation is minimally invasive and, thus, is a reasonable surgical option for patients who often have complex medical histories. Furthermore, it offers hope in increasing limb perfusion to such an extent that a major amputation may be prevented or at least delayed. Extending limb survivorship of this patient population by even a minimum of 3 months improves quality of life. Freedom from MALE was 91.1% (95% CI, 79.9%96.2%) at 3 months and 75.6% (95% CI, 59.4%-86.1%) at 12 months. If difficulty delivering the stem cells was encountered during the procedure, a secondary intervention was performed to improve delivery and to deliver stems cells as distally as possible. It should be noted that this subpopulation of patients experienced a higher incidence of major amputations compared with the overall population, but it is challenging to predetermine when such intraoperative difficulties will be encountered. For this reason and because this is a limb salvage procedure, the patient selection criteria for the procedure has not been modified. Similar BM-MNC implantation studies involving intramuscular injection in patients with severe PAD or chronic limb ischemia have reported limb salvage rates of 53% to 86.3% at 1 year.10,21,22 Although these studies involved patient populations with similar ABIs (range, 0.22-0.33) compared with our study,10,21,22 the study by Higashi et al10 was the only one that reported a similar baseline ulceration rate (>90%). The baseline ulcerations reported by Murphy et al22 and Amann et al22 were much lower, at 31% and 74.5%, respectively. A dual injection technique was chosen to increase cell invasion to the targeted areas intra-arterially and to maximize stem cell delivery locally to ischemic areas.16,17,23,24
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Fig 2. Kaplan-Meier survivorship curve for the bone marrow mononuclear cell (BM-MNC) implantation procedure using freedom from (a) major amputation, (b) postoperative death, and (c) major amputation and postoperative death as survival end points. Because no major vascular reinterventions were performed after BM-MNC implantation, the survival curve for freedom from amputation also represents major above-ankle amputation survivorship.
The intra-arterial injection delivers stem cells to targeted vessels in an antegrade fashion, which directs a high concentration of stem cells to ischemic muscle regions that still are perfused.23 The intramuscular injections at several sites locally disperses a high concentration of stem cells directly into the ischemic musculature. Exclusive use of intra-muscular injections, particularly when confined to the calf, may not affect regions surrounding the femoropopliteal tract or pedal arteries, which often are compromised in patients with advanced, multilevel PAD and may be accessed by intra-arterial injections.24 Conversely, local intramuscular injections are ideal for patients with superficial femoral artery occlusions that may prevent stem cells injected intra-arterially from reaching ischemic muscles.23
The clinical improvements observed in the current study and two previous studies23,24 show this technique of promoting neoangiogenesis through dual physiologic pathways appears effective and should be studied in more detail. Study strengths include complete 3-month follow-up for the study population and continued monitoring of limb status. The main limitation of this study is that a control group was not used. However, because the target patient population for this procedure had end-stage disease and amputation was the only recommended alternative, the senior author did not believe it was ethical to withhold a treatment that offered the possibility of limb salvage. Visual confirmation of neoangiogenesis is difficult given the inherent variability in technique and diagnostic
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imaging protocols. Because of this challenge and also due to the significant comorbidities associated with end-stage PAD patients, angiography was not included as part of the follow-up evaluation. Further study, including endothelial surface marker testing and tissue mitogen assays, is needed to better understand the physiologic response to BM-MNC implantation. Because such testing was beyond the scope of this interventional study, analysis of cell types was added to the study protocol to provide some preliminary data regarding the BM aspirate content. CONCLUSIONS The goal of this dual BM-MNC implantation technique was to improve tissue perfusion to salvage limbs in patients with severe PAD for whom amputation was the only recommended treatment option. The final analysis of our study demonstrates that the procedure effectively delayed or prevented major amputation in the target patient population and, therefore, should be considered as a limb salvage strategy in patients with severe limbthreatening PAD. We acknowledge Alan Parks, DO, Alegent Creighton Health Bergan Mercy Medical Center, Omaha, Nebraska, for contributing to the concept and design of the study and data acquisition, and Jason D. Johnson, DO, Fellow, Plastic and Reconstructive Surgery, Philadelphia College of Osteopathic Medicine, Philadelphia, Pennsylvania, for contributing to data acquisition. AUTHOR CONTRIBUTIONS Conception and design: RF, KS, TH Analysis and interpretation: RF, KS, RP, TH, JH, MW Data collection: RF, TH, JH, MW Writing the article: RF, JH, MW Critical revision of the article: RF, KS, RP, TH, JH, MW Final approval of the article: RF, KS, RP, TH, JH, MW Statistical analysis: MW Obtained funding: Not applicable Overall responsibility: RF REFERENCES 1. Attanasio S, Snell J. Therapeutic angiogenesis in the management of critical limb ischemia: current concepts and review. Cardiol Rev 2009;17:115-20. 2. Fadini GP, Agostini C, Sartore S, Avogaro A. Endothelial progenitor cells in the natural history of atherosclerosis. Atherosclerosis 2007;194: 46-54. 3. De Vries M, Ouwendijk R, Kessels AG, de Haan MW, Flobbe K, Hunink MG, et al. Comparison of generic and disease-specific questionnaires for the assessment of quality of life in patients with peripheral arterial disease. J Vasc Surg 2005;41:261-8. 4. Feinglass J, Pearce WH, Martin GJ, Gibbs J, Cowper D, Sorensen M, et al. Postoperative and late survival outcomes after major amputation: findings from the Department of Veterans Affairs National Surgical Quality Improvement Program. Surgery 2001;130:21-9.
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Submitted Dec 15, 2014; accepted Feb 13, 2015.