Cell therapy, a new standard in management of chronic critical limb ischemia and foot ulcer

Abstract

Fifty percent of diabetics (7% of general population) suffer from peripheral arterial occlusive disease, which may lead to amputation due to critical limb ischemia (CLI). The aim of our study was to prevent major limb amputation (MLA) in this group of patients using a local application of autologous bone marrow stem cells (ABMSC) concentrate. A total of 96 patients with CLI and foot ulcer (FU) were randomized into groups I and II. Patients in group I (n = 42, 36 males, 6 females, 66.2 ± 10.6 years) underwent local treatment with ABMSC while those in group II (n = 54, control, 42 males, 12 females, 64.1 ± 8.6 years) received standard medical care. The frequency of major limb amputation in groups I and II was 21% and 44% within the 120 days of follow up, respectively (p < 0.05). Only in salvaged limbs of group I both toe pressure and toe brachial index increased (from 22.66 ± 5.32 to 25.63 ± 4.75 mmHg and from 0.14 ± 0.03 to 0.17 ± 0.03, respectively, mean ± SEM). The CD34(+) cell counts in bone marrow concentrate (BMC) decreased (correlation, p = 0.024) with age, even though there was no correlation between age and healing. An unexpected finding was made of relative, bone marrow lymphopenia in the initial bone marrow concentrates in patients who failed ABMSC therapy (21% of MLA). This difference was statistically significant (p < 0.040). We conclude ABMSC therapy results in 79% limb salvage in patients suffering from CLI and FU. In the remaining 21% lymphopenia and thrombocytopenia were identified as potential causative factors, suggesting that at least a partial correction with platelet supplementation may be beneficial.

Figure 1

CD34⁺ counts decrease with age (A) but the decrease in CD34⁺ cell count does not correlate with wound nonhealing (B). A significant correlation between decreasing CD34⁺ in BMA and advancing age was found and was consistent with the previously published data on SC aging and SC count lowering with age (Spearman’s rho = −0.348, p = 0.024). Yet, we did not find the often extrapolated effect on healing. There was no statistical difference between the healing and nonhealing group and CD34⁺ counts (p = 0.873) in group I. We observed one extreme outlayer (marked as *¹² in B).

Figure 2

Lymphopenia in the initial BMC was evident in patients who did not heal despite ABMSC transplantation. Only patients with significant baseline lymphopenia and thrombocytopenia (p < 0.05) in the bone marrow aspirate and concentrate went on to a major limb amputation (21% of group 1), compared to healed patients (79%) (A). The low lymphocyte counts were not due to the underlying diabetes (B) and represented a distinct immune defect. We observed one extreme outlayer marked as *² and outlayers o¹⁹ and o² with higher lymphocytes count.

Figure 3

Correlation between number of CD34⁺ cells and number of platelets in bone marrow concentrate. A comparison of a subgroup of patients treated with ABMSCs that healed versus those that did not revealed a significant direct monotonous (nonlinear) dependence of platelets on CD34⁺ cell concentration; that is, a higher CD34⁺ cell concentrations correspond to higher concentration of platelets (Spearman’s rho = 0.679, p = 0.006) (B). In addition, the majority of patients that did not heal clustered in lower left corner, indicating low platelet and CD34⁺ cells. In contrast, the group of patients that healed showed a wide distribution of relative ratios of platelets and CD34⁺ cells (A).

Figure 4

Patients with significant lymphopenia, thrombocytopenia, and low levels of VEGF in BMC did not respond well to ABMSC therapy. All limbs in this patient population were amputated (21%) within 120 days after ABMSC treatment. As evident, the combination of low platelet counts and low VEGF levels is associated with poor healing (A). Statistically significant correlation (r = −0.549, p = 0.010) only exists between the number of platelets and VEGF level in the group of patients treated with ABMSCs who had improved circulation and healed, but not in those without healing (r = −0.324, p = 0.434) (B).

Figure 5

Representative photographs of limb ulcers before and 90 days after ABMSC therapy. During 90 days after ABMSC therapy all ulcers associated with CLI healed in those patients (100%) with normal lymphocyte and thrombocyte counts in BMC at the time of procedure.

Figure 6

Representative angiography of limbs before and 120 days after the ABMSC therapy. Follow-up angiography reveals clear areas of neovascularization and collateral vessel formation along the calf vessels at the day 120 following administration of ABMSCs. The images correspond to the patients presented in Figure 5 with healed wounds.