Lower limb arterial bypass surgery is an effective modality for the treatment of lower limb ischemia. But bypass surgery may not be possible when distal runoff vessels are poor or when general conditions of the patients are poor. Recently, there have been some successful reports using vascular progenitor cells from autologous bone marrow cell implantation.1–3 To explore the effect of transplantation of autologous bone marrow mononuclear cells (BMMCs) on the treatment of lower limb ischemia and to compare the effect of intra-arterial transplantation with that of intra-muscular transplantation. We report our experience with the treatment of 35 severe ischemic lower limbs (32 patients) by transplantation of autologous BMMCs.
Thirty-two patients (35 limbs) who were admitted into the Department of Vascular Surgery of Xuanwu Hospital in Beijing for treating chronic ischemic lower limbs from March 2003 to April 2004 were studied. Of these patients, 20 were male, and 12 were female, with 21 right legs and 14 left legs affected. The mean age of the patients was 69.5 years (51–82 years old). Diabetic foot was found in 32 limbs (29 patients), failed lower limb arterial bypass in 2 limbs (2 patients), and Burger's disease in 1 limb. In 29 diabetic patients, the history of the disease ranged from 3 months to 25 years.
The 32 patients with lower limb ischemia were divided into two groups by randomization. Eighteen limbs of 16 patients received local intra-muscular injection of autologous bone marrow mononuclear cells (group 1) and 17 limbs of 16 patients received intra-arterial injection of autologous bone marrow mononuclear cells (group 2).
Clinical symptoms, physical examinations and other vascular assessments
We used a 5-level scale to evaluate the severity of lower limb pain: level 0: no pain (3 limbs); level 1: occasional pain only remembered by the patients when they were asked (3 limbs); level 2: frequent pain which could be controlled with or without occasional analgesic (6 limbs); level 3: frequent use of analgesic (11 limbs); and level 4: sleep-disturbing pain on which common analgesic did not relief the pain (12 limbs).
Among the 32 limbs with rest pain, 20 limbs had foot pain, 12 had foot and calf pain. There were 17 limbs and 15 limbs with rest pain in group 1 and group 2 respectively. Eight had leg pain accompanied by numbness. Among the 3 limbs without rest pain, 2 limbs of 2 patients were insensible to their hosts after cerebral infarction; another one suffered from intermittent claudication.
Sense of coldness
We used a 5-level scale to evaluate the sense of coldness of the lower limbs: level 0: no sense of coldness (5 limbs); level 1: a sense of occasional coldness (1 limb); level 2: a sense of frequent coldness (8 limbs); level 3: a dramatic sense of coldness in the limb and a certain degree of relief to be reached by local heat preservation (12 limbs); and level 4: a dramatic sense of coldness in the limb and no relief to be reached by local heat preservation (9 limbs). A total of 30 limbs had different degrees of cold sensation.
We used a 5-level scale to evaluate the severity of lower limb intermittent claudication. When walking at a speed of 60–70 m/min, the severity of lower limb claudication was defined as follows: level 0: claudication distance greater than 500 meters; level 1: claudication distance between 300 and 499 meters; level 2: claudication distance between 100 and 299 meters; level 3: claudication distance between 10 and 99 meters (1 limb, 50 meters); and level 4: rest pain and claudication distance less than 10 meters (32 limbs).
Improvement of symptom
Therapeutic efficacy was evaluated by comparing rest pain, sense of coldness, and intermittent claudication before and after the procedure. We used different terms to determine the levels of improvement. “Disappearance” was defined as each of the symptoms reached level 0; “improvement” was defined as each of the symptoms improved by one level; “marked improvement” was defined as each of the symptoms improved by 2–3 levels; and “no change” was defined as no improvement in these symptoms.
Other vascular assessments
Ankle/brachial index (ABI): all patients had the ABI test before the procedure. Eighteen limbs had an ABI of 0, 6 limbs between 0.1 and 0.3, 8 limbs between 0.31 and 0.5, and 3 limbs between 0.51 and 0.8.
Transcutaneous oxygen pressure (tcPO2): all patients had medial ankle tcPO2 test before the procedure. tcPO2 was between 0 and 10 mmHg in 7 limbs, between 11 and 20 mmHg in 12 limbs, between 21 and 30 mmHg in 11 limbs, and more than 30 mmHg in 5 limbs.
Angiography: a 4-level grading system was employed: 0 (no collateral vessels), +1 (minimal collateral vessels), +2 (moderate collateral vessels), +3 (significant collateral vessels).
Transplantation of autologous BMMCs
Autologous BMMCs were intra-muscularly transplanted into 18 limbs (16 patients). About 300 ml of autologous bone marrow was drawn from the ilium under local anaesthesia. A total of 40 ml bone marrow mononuclear cell suspension was obtained following deposition, centrifugation and separation. Under extramural or lumbar anesthesia and after routine skin preparation, the suspension was injected into the lower limb muscles along the arterial tree using a syringe with a 16 Gauge needle. About 1 ml of the suspension was injected at each site. Injected sites were wrapped after the injection. Among the 18 limbs of the 16 patients, 2 limbs received injection into the thigh and the calf at the same time, 1 limb received thigh injection first, which was followed by calf injection one week later, and the other 15 limbs received calf injection only. The thigh injection was performed in patients with proximal occlusion of the superficial femoral artery demonstrated by angiography.
Autologous BMMCs were intra-arterially transplanted into 17 limbs (16 patients). The procedure for obtaining the autologous bone marrow was similar to the method described above. The only difference was that the needed suspension was 10 ml. Under local anesthesia, a balloon catheter was introduced to the corresponding artery via either ipilateral common femoral artery puncture (antegrade) or retrograde contralateral femoral artery puncture. The balloon was distended for 3–5 minutes to block the blood stream and then the mononuclear cell suspension was injected into the distal artery. Among 17 limbs of the 16 patients, the balloon was used to occlude the common femoral artery or profunda femoris artery in 3 limbs with proximal occlusion of the superficial femoral artery, to occlude the superficial femoral artery in 6 limbs with mid and distal occlusion of the superficial femoral artery, and to occlude the popliteal artery in 8 limbs with occlusion of the calf artery.
Enumeration data employed χ2 test, of which correction formula was used when T value was less than 5. Data were expressed as mean ± standard error of the mean (SE). A P value <0.05 was considered statistically significant. SPSS 10.0 statistical package was applied.
The only case of intermittent claudication in the two groups reached “improvement” with an increase of walking distance from 50 meters to 300 meters after the procedure.
Of the 17 limbs with rest pain in group 1, rest pain disappeared in 12 limbs, improved in 1 limb, and there had no change in 4 limbs. Of the 15 limbs with rest pain in group 2, rest pain disappeared in 13 limbs, improved in 1 limb (this patient died from heart failure two weeks after the procedure, but the pain in the foot was improved before death and there was no change in 1 limb. The effective rate of the two groups in pain relief was 76.5% (13/17) and 93.3% (14/15) respectively. There was no statistically significant difference in the improvement of rest pain between the two groups (P=0.32).
As to the improvement of the sense of coldness, both groups reached 100%. Of the 16 limbs with coldness in group 1, cold sense disappeared in 15 limbs and improved in 1 limb. Of the 14 limbs with coldness in group 2, cold sense disappeared in 13 limbs and improved in 1 limb.
Comparison of limb salvage
Five limbs were amputated at 2, 3, 4, 6, and 7 weeks after the procedure, including 3 amputations below the knees in group 1 and 1 amputation below the knee in group 2. One patient had most part of his foot necrotized before the therapy in group 1. The patient received foot amputation two months after the therapy. The limb salvage rates of the two groups were 83.3% (15/18) and 94.1% (16/17) respectively. There was no statistically significant difference in limb salvage between the two groups (χ2=0.22, P >0.05).
There were 2 deaths (one in each group) within 2 months after the procedure. Both patients died from heart failure. The mortality was 6.25%.
ABI increased in 8 limbs (44.4%) in group 1. There were increases between 0.1 and 0.3 in 2 limbs, between 0.31 and 0.5 in 2 limbs, and of more than 0.5 in 4 limbs; there was no change of ABI in 10 limbs, including 1 death and 4 amputations. ABI increased in 7 limbs (41.2%) in group 2. There were increases between 0.1 and 0.3 in 1 limb, between 0.31 and 0.5 in 3 limbs, and of more than 0.5 in 3 limbs; and there was no change of ABI in 10 limbs, including 1 death and 1 amputation. There was no significant difference between the two groups (P >0.05).
tcPO2 was measured at 1, 2 and 4 weeks after the procedure. The result of week 4 was used as a parameter to evaluate the efficiency of the therapy. In group 1, the tcPO2 increased between 1 and 5 mmHg in 5 limbs, between 5.1 and 10 mmHg in 3 limbs, between 10.1 and 20 mmHg in 2 limbs, between 20.1 and 30 mmHg in 2 limbs, and there was no change in 6 limbs. In group 2, the tcPO2 increased between 1 and 5 mmHg in 4 limbs, between 5.1 and 10 mmHg in 5 limbs, between 10.1 and 20 mmHg in 3 limbs, between 20.1 and 30 mmHg in 2 limbs, and there was no change in 3 limbs. There was no significant difference between the two groups (P >0.05).
Angiography of ischemic limb
Fifteen lower limbs of 14 patients received angiography. The levels of newly formed collateral vessels were +1, +2, +3 in 1, 2, 5 limbs respectively for group 1, and +1, +2, +3 in 1, 2, 4 limbs respectively for group 2. There was no statistically significant difference between the 2 groups (P >0.05).
There are many treatment options such as arterial bypass surgery and balloon angioplasty available for the treatment of lower limb ischemia.4–11 However, it is difficult to treat the patients with poor arterial outflow or with poor general conditions. The effect of medical treatment alone is far from ideal, especially in patients with diabetic foot. A high level amputation is inevitable in these patients.12
The process of new blood vessel growth is frequently referred to as angiogenesis. However, as currently understood, neovascularization is the result of several processes, including angiogenesis, arteriogenesis, and, potentially, vasculogenesis.
The angiogenesis describes the sprouting of new capillaries from postcapillary venules,13 and in adults, it is stimulated mainly by tissue hypoxia via activation of hypoxia-inducible factor (HIF)-1α expression. The arteriogenesis refers to the process of maturation or perhaps de novo growth of collateral conduits.14,15 That are frequently of a sufficient diameter to be visualized angiogenesis.16 The vasculogenesis is the process of an in situ formation of blood vessels from circulating endothelial progenitor cells (EPCs) and vascular progenitor cells.17,18 Therefore, therapeutic neovascularization is an important strategy to salvage tissue from critical ischemia.1,19
Recently, there are some reports on successful use of vascular progenitor cells from autologous bone marrow cell implantation2,3,20 to treat lower limb ischemia. We transplanted autologous bone marrow cells by either intra-muscular injection or intra-arterial injection to randomly treat patients with critical ischemia of the lower limbs. We obtained exciting results from this therapy in some patients.21–23 We found that the rate of rest pain improvement after the procedure was 76.5% and 93.3% in the two groups respectively. There was no statistically significant difference between two approaches. There was improvement of sense of coldness in the patients of both groups after the procedure. The effective rate was 100%. Transplantation of autologous bone marrow mononuclear cells was effective in relieving symptoms of the lower limbs with critical ischemia. Most of the patients avoided amputation or had the levels of their amputation lowered after the therapy. Of the 5 patients who received amputation, one patient showed most part of his foot necrotized before the therapy, but there was no pain in the foot. The patient received amputation 2 months after the therapy. Three patients underwent amputation because of rest pain. However the levels of amputation were lower than what was required without the therapy.12 The aim of lowering the amputation level was achieved. One patient underwent amputation because the ischemia did not improve and the foot infection could not be controlled. Two patients still suffered from rest pain but did not receive amputation. One patient was diagnosed as thromboangiitis obliterans. This patient still suffered from rest pain following lumber sympathectomy 3 weeks after the transplantation. The cause of pain was considered to be dolantin addiction rather than ischemia. One patient's pain was due to a localized foot ulcer and he was still in the process of follow-up and further treatment.
The results of our study showed that following transplantation of autologous bone marrow mononuclear cells, ABI increased in 15 limbs and that the increase was over 0.5 in 7 of these limbs. It is impossible to increase ABI significantly in a short time. The ABI of more than half of the patients did not change after the therapy. A possible explanation is that mononuclear cell transplantation may lead to more capillary formation and result in the formation of small collateral vessels. The ABI would increase only when there is no severe pathological change in the dorsal pedal artery or posterior tibial artery and the blood could flow to these arteries via collateral vessels in the calf. The patient symptoms in our groups were severe and there were many foot necroses and the arteries could not be detected on angiography. This indicates that there may be pathological changes in the dorsal pedal artery or posterior tibial artery. Other possible reasons include more time required for reformation of vessels and more transplantation needed to enhance the clinical effect. It should be emphasized that the patients should have exercise training early and frequently after the procedure, which would be helpful for improvement of ischemia. One patient in our groups began to exercise 6 hours after the procedure and his limb started to warm on the third day and his rest pain disappeared on the fourth day after the procedure. Exercise training may enhance the process of transformation of mononuclear cells into endothelial cells and vasculogenesis of endothelial cells.
Neither of the methods produced 100% clinical efficacy. We believe that it is related to the selection of patients. At the beginning of our study, the cases selected were those who required high-level amputation. Even with those patients, the results are exciting. No patients in our groups had side effects during the therapy. This demonstrates that the therapy is safe. The angiography of 15 lower limbs in 14 patients showed the formation of new collateral vessels. However it should be mentioned that nearly half of the patients did not have angiography. Ankle transcutaneous oxygen pressure in most of the patients was improved to above 20 mmHg. The pressure less than 20 mmHg is one of the clinical indications for amputation.
Transplantation of autologous bone marrow mononuclear cells is not yet a perfect way for treating the critical ischemia of the lower limbs. In our groups, some patients had pain or received amputation after the therapy. However, this technique is one of the effective methods for the treatment of lower limb ischemia, especially in those who could not have arterial bypass graft surgery due to poor run-off or poor general medical conditions.
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