Autologous bone marrow mononuclear cell therapy is safe and promotes amputation-free survival in patients with critical limb ischemia

Abstract

Objective: The purpose of this Phase I open label nonrandomized trial was to assess the safety and efficacy of autologous bone marrow mononuclear cell (ABMNC) therapy in promoting amputation-free survival (AFS) in patients with critical limb ischemia (CLI).

Methods: Between September 2005 and March 2009, 29 patients (30 limbs), with a median age of 66 years (range, 23-84 years; 14 male, 15 female) with CLI were enrolled. Twenty-one limbs presented with rest pain (RP), six with RP and ulceration, and three with ulcer only. All patients were not candidates for surgical bypass due to absence of a patent artery below the knee and/or endovascular approaches to improving perfusion was not possible as determined by an independent vascular surgeon. Patients were treated with an average dose of 1.7 ± 0.7 × 10(9) ABMNC injected intramuscularly in the index limb distal to the anterior tibial tuberosity. The primary safety end point was accumulation of serious adverse events, and the primary efficacy end point was AFS at 1 year. Secondary end points at 12 weeks posttreatment were changes in first toe pressure (FTP), toe-brachial index (TBI), ankle-brachial index (ABI), and transcutaneous oxygen measurements (TcPO(2)). Perfusion of the index limb was measured with positron emission tomography-computed tomography (PET-CT) with intra-arterial infusion of H(2)O(15). RP, using a 10-cm visual analogue scale, quality of life using the VascuQuol questionnaire, and ulcer healing were assessed at each follow-up interval. Subpopulations of endothelial progenitor cells were quantified prior to ABMNC administration using immunocytochemistry and fluorescent-activated cell sorting.

Results: There were two serious adverse events; however, there were no procedure-related deaths. Amputation-free survival at 1 year was 86.3%. There was a significant increase in FTP (10.2 ± 6.2 mm Hg; P = .02) and TBI (0.10 ± 0.05;P = .02) and a trend in improvement in ABI (0.08 ± 0.04; P = .73). Perfusion index by PET-CT H(2)O(15) increased by 19.3 ± 3.1, and RP decreased significantly by 2.2 ± 0.6 cm (P = .02). The VascuQol questionnaire demonstrated significant improvement in quality of life, and three of nine ulcers (33%) healed completely. KDR(+) but not CD34(+) or CD133(+) subpopulations of ABMNC were associated with improvement in limb perfusion.

Conclusion: This Phase I study has demonstrated safety, and the AFS rates suggest efficacy of ABMNC in promoting limb salvage in "no option" CLI. Based on these results, we plan to test the concept that ABMNCs improve AFS at 1 year in a Phase III randomized, double-blinded, multicenter trial.

Copyright © 2011 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.

Figures

Figure 1

Bone marrow aspirate is loaded into a closed sterile 60-mL MarrowStim™ centrifugation tube in the operating room (A) and placed into the centrifuge device where the buffy coat is separated at 1500 rpm for 15 min (B). The ABMNC rich buffy coat is aspirated from the tube (C) and and 0.75 ml. aliquots are delivered via IM injections at 2-cm intervals, 1.5-cm deep into the gastrocnemius muscle, along the medial and lateral aspect of the index limb (D).

Figure 2. Change in FTP, TBI, and ABI from baseline (mean ± SE)

FTP increased by 10.2±6.2 mmHg (P=.02) (A) and TBI increased by 0.10 ± 0.05 (P=.02) (B) at week 12 from baseline. The ABI increased by 0.08 ± 0.04 at week 12 but was not significant (P=.73) (C).

Figure 2. Change in FTP, TBI, and ABI from baseline (mean ± SE)

FTP increased by 10.2±6.2 mmHg (P=.02) (A) and TBI increased by 0.10 ± 0.05 (P=.02) (B) at week 12 from baseline. The ABI increased by 0.08 ± 0.04 at week 12 but was not significant (P=.73) (C).

Figure 2. Change in FTP, TBI, and ABI from baseline (mean ± SE)

FTP increased by 10.2±6.2 mmHg (P=.02) (A) and TBI increased by 0.10 ± 0.05 (P=.02) (B) at week 12 from baseline. The ABI increased by 0.08 ± 0.04 at week 12 but was not significant (P=.73) (C).

Figure 3. Radiolabeled H2O15 Positron Emission Tomogaphy-CT Scan

A representative image of blood perfusion (BP) quantified with intra-arterial injection of H2O15 before ABMC treatment (A) in the left leg and after (B). A Blood Perfusion Index (BPI) was calculated by comparing the ratio of H2O15 peak tracer uptake level of the untreated:treated in the segment of the leg denoted by the dashed line. In this patient the BPI increased from 0.38 at baseline to 0.54 (42%) at 12 weeks , concurrent with an increase in the ABI from 0 to 0.32 in the same period.

Figure 4. Correlation of bone marrow subpopulations and changes in perfusion

There was no correlation between total MNC count and changes in ABI (R2 = 0.028) (A). Using FACS analysis of EPC surface markers in ABMNCs we found a significant difference in KDR+ cells between responders to treatment as compared to non-responders (1.02 ± 0.39% vs. 0.36 ± 0.12 %, P = .008), respectively. There were no differences in CD34+ (4.21± 0.80% vs. 3.97 ± 0.40%, P = .14) or CD133+ (0.59 ± 0.12% vs. 0.68 ± 0.17%, P= .9) subpopulations between responders and nonresponders, respectively.

Figure 4. Correlation of bone marrow subpopulations and changes in perfusion

There was no correlation between total MNC count and changes in ABI (R2 = 0.028) (A). Using FACS analysis of EPC surface markers in ABMNCs we found a significant difference in KDR+ cells between responders to treatment as compared to non-responders (1.02 ± 0.39% vs. 0.36 ± 0.12 %, P = .008), respectively. There were no differences in CD34+ (4.21± 0.80% vs. 3.97 ± 0.40%, P = .14) or CD133+ (0.59 ± 0.12% vs. 0.68 ± 0.17%, P= .9) subpopulations between responders and nonresponders, respectively.

Figure 5. Changes in Rest Pain and Quality of Life

Using a 10-cm visual analog scale RP significantly decreased at 12 weeks by 2.2 ± 0.6 cm. (P=.02) (A). The VascuQol total score increased from 2.62 to 4.07 at 12 weeks (P= .008) (B).

Figure 5. Changes in Rest Pain and Quality of Life

Using a 10-cm visual analog scale RP significantly decreased at 12 weeks by 2.2 ± 0.6 cm. (P=.02) (A). The VascuQol total score increased from 2.62 to 4.07 at 12 weeks (P= .008) (B).

Figure 6. Ulcer healing

Nine patients with ulceration of the index limb were enrolled. Complete healing of the ulcer with re-epithelialization was achieved in three patients (33%) at 12 weeks. Representative photographs are shown of a 46 year old woman with Buerger’s disease and an ulcer on the right first toe at baseline (A1) and at 12 weeks (A2). FTP concurrently increased from 2 to 25 mmHg in this period. A 73 year old male with ulceration of the right heel with exposed calcaneus bone at baseline (B1) and at 12 weeks (B2). There was sufficient growth of granulation tissue to cover the calcaneus and support a split thickness skin graft (STSG). Although not considered a success of trial therapy alone, as a STSG was required for re-epithelialization, - this case is presented to illustrate the potential of ABMNC in promoting wound healing, including via enablement of other procedures such as STSG.