Histological changes after implantation of autologous bone marrow mononuclear cells for chronic critical limb ischemia

Animal experiment models have demonstrated that implantation of autologous bone marrow mononuclear cells (BM-MNC) is an effective technique for inducing therapeutic angiogenesis. Histological studies have shown an increase in the capillary/muscle fibre ratio with incorporation of implanted cells into the vasculature. In humans, clinical improvement has been reported in patients with critical limb ischaemia (CLI). However, few histological data have been yet provided in patients after cell therapy. We had the opportunity to observe histological changes in two patients who required amputation after cell therapy. In October 2004 we initiated a prospective non randomised trial to evaluate the feasibility and efficacy of implanting autologous BM-MNC in patients presenting with CLI related to atherosclerosis obliterans who had no possibility of surgical or endovascular revascularization. The study protocol was approved by the Ethics Committee and written informed consent for participation was obtained from all subjects. BM-MNC were collected as described previously. Briefly, 500ml of bone marrow were collected under general anaesthesia. MNC were separated using a blood-cell separator (COBE SPECTRA, GAMBRO BCT, Lakewood, USA) and concentrated to produce a final volume of 40ml. Cell implantation was performed within 8 h after cell collection, by 30 trans-cutaneous injections of 1ml of cell suspension into the gastrocnemius muscle. Clinical parameters, ankle-brachial pressure index (ABPI), trans-cutaneous oxygen pressure (TcPO2) and biological parameters were then monitored. Patient 1 was a 71-year-old man who presented with CLI secondary to the occlusion of a left femoro-tibial graft implanted 5 years earlier. ABPI and TcPO2 were respectively 0 and 1mm Hg and he had a necrotic big toe, which had been present for 1 month. The cellular concentrate contained 4.6 10 BM-MNC, among which 16.0 10, 11 10 and 0.11 10 were respectively CD34þ , CD34þ / CD133þ and CD34þ /CDF133þ /VEGFR2þ . After implantation, no clinical improvement was observed. Leg amputation was performed on day 23. Patient 2 was a 46year-old man who presented with CLI secondary to the occlusion of a left femoro-tibial graft implanted 5 years before. ABPI and TcPO2 were respectively 0.4 and 1mm Hg and an ulcer of the left foot had been present for 3 weeks. The cellular concentrate contained 8.3 10 BM-MNC among which 45.6 10, 19 10 and 0.07 10 were respectively CD34þ , CD34þ /CD133þ and CD34þ /CDF133þ /VEGFR2þ . Because of clinical deterioration, amputation of the leg was performed on day 44. Both patients were treated with antiagregant medications and statins before and after cell implantation and had ceased smoking. No additional therapeutic measures were undertaken between cell therapy and amputation. After leg amputation, muscle samples were harvested from the gastrocnemius at four levels, in both cases, superior limit of excision (i.e. above the area of cell implantation) and proximal, mid and distal calf. Samples were fixed overnight in 4% formalin, and then processed in paraffin blocks. Three-micrometer sections were stained with hematoxylin–eosin–saffron (HES). Immunostaining was performed on selected blocks using the following antibodies: CD3, CD20, CD31, CD34, CD68, CD79a, CD117 (c-kit), Willebrand factor, actin, desmin and myeloperoxidase (MPO). Three legs amputated from CLI patients treated by the same team without cell therapy, were used as controls. On HES slides, muscle histology was normal at the upper limit of muscle resection. Conversely, prominent oedema was observed in the fascicles and in the inter-fascicular compartment of the lower sites. Oedema was more pronounced in the middle and distal parts of the muscle and was associated with shrinkage of muscle fibres and with large numbers of capillaries running along the side of muscle fibres. Two particular kinds of change were observed. First, in the peri-fascicular compartments, a