A Multicenter Prospective Interventional Trial of Therapeutic Angiogenesis Using Bone Marrow-Derived Mononuclear Cell Implantation for Patients With Critical Limb-Threatening Ischemia Caused by Thromboangiitis Obliterans

Abstract

Background: Thromboangiitis obliterans (TAO) can lead to the development of critical limb-threatening ischemia (CLTI). Despite conventional treatments, such as smoking cessation or revascularization, young patients (<50 years) still require limb amputation. Therapeutic angiogenesis using bone marrow-derived mononuclear cell (BM-MNC) implantation has been tested and shown to have reasonable efficacy in CLTI. In this multicenter prospective clinical trial, we evaluated the safety and efficacy of BM-MNC implantation in CLTI patients with TAO.

Methods and Results: We enrolled 22 CLTI patients with skin perfusion pressure (SPP) <30 mmHg. The primary endpoint of this trial is the recovery of SPP in the treated limb after a 180-day follow-up period. Secondary endpoints include the pain scale score and transcutaneous oxygen pressure (TcPO₂). One patient dropped out during follow-up, leaving 21 patients (mean age 48 years, 90.5% male, Fontaine Class IV) for analysis. BM-MNC implantation caused no serious adverse events and increased SPP by 1.5-fold compared with baseline. Surprisingly, this effect was sustained over the longer term at 180 days. Secondary endpoints also supported the efficacy of this novel therapy in relieving pain and increasing TcPO₂. Major amputation-free and overall survival probabilities at 3 years among all enrolled patients were high (95.5% and 89.5%, respectively).

Conclusions: BM-MNC implantation showed safety and significant efficacy in CLTI patients with TAO.

Thromboangiitis obliterans (TAO; Buerger’s disease) is more common among men in their 30s and 40s, and shows a strong causal association with smoking. However, the pathogenesis of TAO remains unknown, and it is categorized as an incurable disease.¹ Approximately 70% of patients with TAO develop critical limb-threatening ischemia (CLTI) of Fontaine Class III or higher during progression, and approximately 10% of these patients undergo major amputation surgery.² Further, approximately 20% of patients require minor amputations of the toes or fingers.^3–5 In addition, approximately 40% of patients with TAO experience work restrictions because of pain at rest, ulcers, and gangrene, and more than 50% of patients experience lifestyle restrictions, which have major impacts on activities of daily living and quality of life.⁶

Smoking cessation and drug and exercise therapies are generally recommended as the standard of care for patients with TAO. Revascularization using bypass surgery and endovascular therapy (EVT) is required for patients with CLTI.^6,7 However, EVT and bypass surgery are difficult in many patients because of the vascular properties or vascular bed. The chronic phase of bypass surgery has a low patency rate, and there is also insufficient evidence for the benefits of EVT because it often exhibits early recoil or reocclusion.^8,9 Therefore, several CLTI patients with TAO are refractory to conventional standard treatments, including revascularization (patients with no-option CLTI).

Endothelial progenitor cells derived from the bone marrow reportedly promote angiogenesis.^10–12 Therapeutic angiogenesis has been introduced clinically for patients with no-option CLTI to improve peripheral blood flow. Several clinical trials, such as the Japan Trial for Therapeutic Angiogenesis using Cell Transplantation, have reported on the safety and efficacy of therapeutic angiogenesis using autologous bone marrow-derived mononuclear cell (BM-MNC) implantation in no-option CLTI patients, particularly those with TAO.^13–20

We designed a multicenter prospective clinical trial to further evaluate the safety and efficacy of BM-MNC implantation for CLTI patients with TAO to develop this novel therapy as an alternative future option for no-option CLTI patients.

Methods

Trial Design

We performed a multicenter prospective interventional trial under Advanced Medical Care Program B. The Kyoto Prefectural University of Medicine was the supervisory research institution, and another 5 institutions agreed to participate in the trial. We enrolled no-option CLTI patients with TAO, who underwent BM-MNC implantation within 28 days of enrollment. We measured all specified parameters on Days 1, 7, 30, 90, and 180 after implantation. The protocol therapy was completed 180 days after BM-MNC implantation. The outcome survey of all enrolled patients was performed within 1 year of the last enrollment. The trial was terminated after completion of the outcome survey.

Participants

The study participants were patients with severe ischemic limbs previously diagnosed with TAO or recently diagnosed with TAO according to Shionoya’s diagnostic criteria as follows: (1) onset at age <50 years; (2) smoking history; (3) lower leg arterial occlusion; (4) the presence or a history of upper limb arterial occlusion or migratory phlebitis; and (5) no risk factors for obstructive arteriosclerosis other than smoking.¹ Participants who met all criteria were diagnosed with TAO; those who met only 4 criteria were diagnosed with subtype TAO.

Further, participants had to meet the following inclusion criteria: (1) male or female aged between 20 and 80 years; (2) TAO and Fontaine Class III or IV; (3) skin perfusion pressure (SPP) of the target limb <30 mmHg during registration; (4) refractory disease or not indicated for conventional standard treatments, such as drug therapy (vasodilators and/or antiplatelet agents and/or analgesics), exercise therapy, sympathetic ganglion block, and revascularization (EVT or bypass surgery); and (5) able to provide written consent to participate in this trial after receiving sufficient explanation from the informed consent form.

The exclusion criteria were as follows: (1) refusal to provide informed consent or being considered unsuitable for the trial because of emotional considerations, even if the disease and procedure were suitable; (2) CLTI in both lower limbs and requiring immediate treatment; (3) life expectancy <1 year because of another comorbid condition; (4) a diagnosis of malignant tumor; (5) ischemic heart disease that has not been treated with revascularization; (6) untreated severe diabetic retinopathy; (7) serious infection, serious hepatic impairment or renal impairment (excluding chronic maintenance dialysis patients), and serious hematological disorders (e.g., leukopenia or thrombocytopenia); (8) severe anemia necessitating blood transfusion, pregnancy, potential pregnancy, or breastfeeding; (9) participation in clinical trials of a drug or medical device simultaneously or within 30 days before enrollment in this trial; and (10) other severe acute, chronic medical, or psychiatric conditions or abnormal clinical test results for which participation in the trial may result in increased risk or may affect the interpretation of our results.

Protocol Procedures

BM-MNC implantation was performed under general anesthesia. First, bone marrow fluid was collected from both iliac bones. The bone marrow fluid collected was then separated by a blood component separator and concentrated to approximately 40–80 mL, comprising >0.5×10⁹ BM-MNCs. The BM-MNCs were evenly injected into the skeletal muscles of the target leg below the knee. After implantation, patients were discharged from hospital on Day 7 and were followed up regularly in the outpatient clinic until Day 180 after implantation. During the follow-up period, vasodilators, antiplatelet agents, and analgesics could be reduced or discontinued; however, they could not be changed or increased. In addition, concomitant drug use in other clinical trials was prohibited, and pain relief therapy using nerve blocks, such as a sympathetic ganglion block and epidural block, could not be performed.

Outcome Measures

The primary endpoint was the change in SPP from the time of enrollment to 180 days after BM-MNC implantation. We measured SPP on the dorsal side of the affected target limb and the proximal site (dorsal or plantar) of the ischemic ulceration of the affected target limb for patients with Fontaine Class III and IV, respectively. In addition, we recorded the measurement site of SPP at enrollment to avoid changes in the measurement site before and after BM-MNC implantation; SPP after BM-MNCs implantation was measured at a similar site to that used at enrollment.

Secondary endpoints were as follows: (1) changes in the 6-min walking distance from enrollment to Day 180; (2) changes in the degree of pain based on the numerical rating scale from enrollment to Day 180; (3) changes in the arterial vascular volume below the popliteal artery of the affected limb by contrast-enhanced computed tomography (CT) from enrollment to Day 180; (4) changes in the ankle-brachial index (ABI) and transcutaneous oxygen pressure (TcPO₂) from enrollment to Day 180; (5) achievement of SPP ≥30 mmHg on Day 180; (6) at least a 30% reduction in the ischemic ulceration area on Day 180; (7) changes in the Fontaine classification from enrollment to Day 180; and (8) the period until major amputation of the affected target limb. A third-party specialist evaluated improvement of the arterial vascular volume below the popliteal artery on contrast-enhanced CT and ischemic ulceration area as a central evaluation. For safety evaluation, the occurrence or severity of adverse events and overall survival were assessed during the trial.

Ethical Considerations

This trial was approved by the Ministry of Health, Labour and Welfare after approval by the Certified Committee for Regenerative Medicine in the Kyoto Prefectural University of Medicine in 2017 (ID: NA8150008). Furthermore, the trial was registered again with the Japan Registry of Clinical Trials (jRCT) in 2019 (ID: jRCTb050190082). It was also registered with the University Hospital Medical Information Network (UMIN) Clinical Trials Registry (ID: UMIN000027383). In accordance with the Helsinki Declaration, written informed consent was obtained from all patients.

Data Collection

We created case report forms (CRFs) to collect information about participants’ baseline and procedural characteristics, evaluation measurements, adverse events, and clinical outcomes from registration to Day 180. Furthermore, we created CRFs for the outcome survey after the trial terms. All data were stored at the Clinical and Translational Research Center, University Hospital, Kyoto Prefectural University of Medicine, Kyoto, Japan. These data were confirmed and fixed by a third-party monitoring officer, based on the source documents and electronic medical records. Subsequently, this trial was independently analyzed by biostatisticians.

Statistical Analyses

The sample size calculation was based on reports by Tateishi-Yuyama et al¹² and Matoba et al.¹⁶ Regarding changes in TcPO₂ values, which can be considered similar to SPP values, the mean±SD was 18±11 and 16.6±9.9 mmHg for Groups A and B, respectively, in the report of Tateishi-Yuyama et al,¹² and approximately 15 mmHg (SD not stated) in the report of Matoba et al.¹⁶ We estimated the mean and SD of the expected SPP change to be 10 and 11 mmHg, respectively. We considered the conservative SPP values were 20 and 0 mmHg if they were below the detection limit at baseline and at the 6-month measurement, respectively. While performing the Wilcoxon signed-rank test at a 1-sided significance level of 2.5%, the required sample size was calculated to be 19, assuming a power of 95%. Considering the excluded cases, the target sample size was set to 25.

The population of participants who excluded protocol violations or discontinued cases among all participants was defined as the full analysis set (FAS). The population of participants who received the protocol treatment was defined as the safety analysis set (SAS). The efficacy evaluation of BM-MNC implantation was analyzed against the FAS. The safety evaluation of BM-MNC implantation was analyzed using the SAS.

All continuous variables are presented as the mean±SD or median and interquartile range, as appropriate for the data distribution; all categorical variables are presented as numbers and percentages. Primary and secondary endpoints with continuous outcome measures were evaluated using the Wilcoxon signed-rank test and the 95% confidence intervals (CIs) of the mean of the change were calculated based on the t-distribution. We performed the time-to-event analysis using the Kaplan-Meier method. For the primary endpoint, we conducted subgroup analyses according to baseline SPP (<20 vs. ≥20 mmHg) and baseline Fontaine classification (III vs. IV). For all analyses, P values were 1-sided, and the significance level was set at 1-sided 2.5%. All statistical analyses were performed using SAS^® 9.4 (SAS Institute, Cary, NC, USA).

Results

Study Flowchart

Twenty-two CLTI patients with TAO underwent BM-MNC implantation in 6 institutions between October 2017 and September 2021. One patient developed sepsis caused by a severe infection of the affected toes, thus leading to amputation 45 days after implantation. The patient subsequently died by suicide 52 days after implantation. Therefore, the protocol therapy was discontinued. Eventually, we analyzed 21 patients who completed the protocol term as the FAS and all 22 enrolled patients as the SAS (Figure 1).

Figure 1.

Flowchart depicting participant selection. BM-MNCs, bone marrow-derived mononuclear cells.

Baseline and Procedural Characteristics

Table 1 summarizes the baseline and procedural characteristics of the 21 patients. The mean age was 47.6 years, and 90.5% were male. The mean age at the first onset of TAO was 39.3 years. All patients had a history of smoking. The complete success rate of smoking cessation at enrollment was 57.1%. A history of drug therapy, exercise therapy, sympathetic ganglion block, EVT, and bypass surgery within 3 years before enrollment was observed in 95.2%, 85.7%, 4.8%, 38.1%, and 4.8% of patients, respectively. The SPP of all affected target limbs at enrollment was <30 mmHg, and the mean SPP was 23.7 mmHg; 57.1% of patients were classified as Fontaine Class IV. The procedure success rate was 100%, and the median number of BM-MNCs implanted in the target-affected limb was 2.6×10⁹ (Table 1).

Table 1. Baseline and Procedural Characteristics in All Patients (n=21)

Age (years)	47.6±12.6
Age at first onset (years)	39.3±12.2
Male sex	19 (90.5)
Height (cm)	168.1±5.4
Weight (kg)	67.7±11.2
BMI (kg/m2)	23.9±3.8
Past smoking	21 (100)
Past illness history
Hypertension	5 (23.8)
Hyperlipidemia	8 (38.1)
Diabetes	3 (14.3)
Cardiovascular diseases	0 (0)
Past treatment history within 3 years before enrollment
Success of smoking cessation	12 (57.1)
Drug therapy	20 (95.2)
Exercise therapy	18 (85.7)
Sympathetic ganglion block	1 (4.8)
Endovascular treatment	8 (38.1)
Bypass surgery	1 (4.8)
LDL apheresis	0 (0)
Hyperbaric oxygen therapy	1 (4.8)
Blood examinations
White blood cell count (×103/μL)	7.7 [6.6–8.7]
Hemoglobin (g/ml)	14.3 [13.1–14.7]
Platelets (×103/μL)	317.0 [261.0–355.0]
C-reactive protein (mg/dL)	0.11 [0.06–0.26]
Total protein (g/dL)	7.1 [6.8–7.2]
Albumin (g/dL)	4.3 [3.9–4.5]
Creatinine (mg/dL)	0.9 [0.7–1.0]
LDL (mg/dL)	113.0 [85.0–131.0]
HbA1c (%)	5.8 [5.4–6.1]
Baseline characteristics of the target limb
Target limb
Right lower limb	9 (42.9)
Left lower limb	12 (57.1)
Fontaine classification
Class III	9 (42.9)
Class IV	12 (57.1)
Skin perfusion pressure value (mmHg)	23.7±4.2
Transcutaneous oxygen pressure value (mmHg)	29.1±14.2
Ankle brachial pressure index	0.8±0.3
6-min walking distance (m)	403.8±160.3
Ulcer size (cm2)	4.4±4.8
Numerical rating scale	5.1±2.0
Vascular volume below the popliteal artery (mL)	38.2±27.6
Procedure of BM-MNC implantation
Procedural success	21 (100)
Total no. BM-MNCs (×109)	2.6 [1.9–2.9]
Total no. CD34-positive cells (×107)	5.6 [3.6–7.0]

Data are presented as n (%), mean±SD, or the median [interquartile range]. BMI, body mass index; BM-MNCs, bone marrow-derived mononuclear cells; LDL, low-density lipoprotein.

Efficacy of BM-MNC Implantation in Patients With TAO

We evaluated the efficacy of BM-MNC implantation in 21 patients with TAO as the FAS. The mean of the primary endpoint, namely the change of mean SPP value from the time of enrollment to Day 180: 26.5 mmHg mean SPP at the time of enrollment: 23.7 mmHg, mean SPP at Day 180: 50.1 mmHg, with a significant improvement in SPP (95% CI 19.5–33.5; P<0.001; Figure 2A). Twenty patients achieved an SPP ≥30 mmHg at 180 days after implantation. Subgroup analyses were performed for the primary endpoint according to baseline SPP (<20 mmHg vs. ≥20 mmHg; Figure 2B,C) or baseline Fontaine classification (Class II vs. Class IV; Figure 2D,E). In the group with SPP <20 mmHg at baseline, there was a tendency for an improvement in SPP on Day 180 (95% CI 3.0–52.6, P=0.031; Figure 2B). In the group with SPP ≥20 mmHg at baseline, there was a significant improvement in SPP on Day 180 (95% CI 18.3–33.8; P<0.001; Figure 2C). In addition, there were significant improvements in SPP on Day 180 compared with baseline in both the Fontaine Class III and Class IV groups (95% CI 13.9–35.9 [P=0.002] and 95% CI 17.1–38.3 [P<0.001], respectively; Figure 2D,E). Regardless of baseline SPP and Fontaine classification, SPP tended to improve after BM-MNC implantation.

Figure 2.

Improvement in the primary endpoint, namely skin perfusion pressure (SPP), during follow-up. (A) Improvement in SPP from enrollment to 180 days after bone marrow-derived mononuclear cell (BM-MNC) implantation. (B,C) Subgroup analysis for the primary endpoint according to baseline SPP values: <20 mmHg (B) vs. ≥20 mmHg (C). (D,E) Subgroup analysis for the primary endpoint according to baseline Fontaine classification: Class III (D) vs. Class IV (E). The boxes show the interquartile range, with the median value indicated by the horizontal line; whiskers show the range and “+” symbols indicate mean values. Outliers are shown as black circles. *P<0.025, ^†P<0.001 compared with baseline.

With regard to secondary endpoints, there were significant improvements on Day 180 compared with baseline in the mean values of numerical rating scale scores (−4.0; from 5.1 to 1.1 [from mean numerical rating scale score at the time of enrollment to mean numerical rating scale score at Day 180]; 95% CI −5.2, −2.9; P<0.001; Figure 3B), arterial vascular volume below the popliteal artery as determined by contrast-enhanced CT (7.7 mL; from 38.2 to 45.9 mL [from mean arterial vascular volume at the time of enrollment to mean arterial vascular volume at Day 180]; 95% CI 5.5–9.9; P<0.001; Figure 3C), and TcPO₂ (14.2 mmHg; from 29.1 to 43.3 mmHg [from mean TcPO₂ value at the time of enrollment to mean TcPO₂ value at Day 180]; 95% CI 5.5–22.9, P=0.002; Figure 3E). The mean change from baseline to Day 180 in 6-min walking distance was 54.3 m (from 403.8 to 458.1 m [from mean 6-min walking distance at the time of enrollment to mean 6-min walking distance at Day 180]), with a tendency for an improvement on Day 180 (95% CI –3.0, 111.6; P=0.049; Figure 3A). In contrast, there was no significant change in the ABI on Day 180 compared with baseline (95% CI −0.04, 0.12; P=0.19; Figure 3D). Among patients with Fontaine Class IV, the ischemic ulceration area improved on Day 180 compared with baseline (4.4 vs. 1.7 cm²), with 80% of patients demonstrating at least a 30% reduction in the area of ischemic ulceration (Figure 3F).

Figure 3.

Improvements in secondary endpoints during the follow-up period after bone marrow-derived mononuclear cell (BM-MNC) implantation: (A) 6-min walking distance; (B) numerical rating scale; (C) vascular volume below the popliteal artery of the affected target limb; (D) ankle-brachial index (ABI); (E) transcutaneous oxygen pressure (TcPO₂); (F) area of ischemic ulceration of the affected target limb. The boxes show the interquartile range, with the median value indicated by the horizontal line; whiskers show the range and “+” symbols indicate mean values. Outliers are shown as black circles. *P<0.025, ^†P<0.001 compared with baseline.

Table 2 summarizes changes in the Fontaine classification from enrollment to Day 180. Of the 12 patients with Fontaine Class IV at enrollment, almost all patients had a reduction in the area of ischemic ulceration area on Day 180, but no change in the Fontaine classification; 1 patient improved to Fontaine Class I. Of the 9 patients with Fontaine Class III at enrollment, 4 patients improved to Fontaine Class II, and 5 patients improved to Fontaine Class I (Table 2).

Table 2. Changes in the Fontaine Classification From Enrollment to Day 180

Enrollment	Day 180
Fontaine IV (n=12)	Fontaine IV (n=11)
	Fontaine III (n=0)
	Fontaine II (n=0)
	Fontaine I (n=1)
Fontaine III (n=9)	Fontaine IV (n=0)
	Fontaine III (n=0)
	Fontaine II (n=4)
	Fontaine I (n=5)

The major amputation-free probability was 100% among the FAS (Figure 4). Conversely, the major amputation-free probability at 1 and 3 years among the SAS was 95.5% and 95.5%, respectively (Figure 5A). According to these results, the long-term major amputation-free probability was maintained after BM-MNC implantation.

Figure 4.

Kaplan-Meier analysis of freedom from major amputation of the affected target limb in the full analysis set. BM-MNCs, bone marrow-derived mononuclear cells.

Figure 5.

Kaplan-Meier analysis of (A) freedom from major amputation of the affected target limb and (B) overall survival in the safety analysis set. BM-MNCs, bone marrow-derived mononuclear cells.

Safety of BM-MNC Implantation in Patients With TAO

We evaluated the safety of BM-MNC implantation in 22 patients with TAO and the SAS. Overall survival probability at 1 and 3 years was 95.5% and 89.5%, respectively (Figure 5B).

Table 3 summarizes clinical outcomes and clinically relevant adverse events after implantation during the trial. One patient underwent a major amputation because of severe wound infection and sepsis 45 days after BM-MNC implantation; this patient died by suicide 52 days after BM-MNC implantation. Another patient died of renal failure 585 days after BM-MNC implantation. In addition, 1 patient underwent a scheduled minor amputation after BM-MNC implantation (Table 3). Serious adverse events occurred in 9.1% of patients. These serious adverse events were not related to BM-MNC implantation. There were no cardiovascular events. Frequent adverse events were temporary increases or decreases in blood examination values from reference ranges (90.9%), pain in the implanted limb (81.8%), gastrointestinal disorders (31.8%), fever (22.7%), limb swelling (18.2%), hypertension (13.6%), and tachycardia (9.1%). In addition, we observed anemia requiring blood transplantation in 9.1% of patients. Most events were non-serious adverse events that occurred because of the surgical invasion associated with BM-MNC implantation or general anesthesia, occurred within 7 days after implantation, and spontaneously improved by the time of discharge.

Table 3. Clinical Outcomes After Bone Marrow-Derived Mononuclear Cell Implantation in All Patients (n=21)

Clinical outcome	No. patients (%)
Death	2 (9.1)
Cause of death
Suicide	1 (4.5)
Renal failure	1 (4.5)
Amputation
Major amputation	1 (4.5)
Minor amputation	1 (4.5)
Cardiovascular events	0 (0)
All-cause adverse events
Serious adverse events	2 (9.1)
Wound severe infection	1 (4.5)
Sepsis	1 (4.5)
Suicide	1 (4.5)
Renal failure	1 (4.5)
Non-serious adverse events	20 (90.9)
Abnormal blood examination values	20 (90.9)
Decrease in albumin	18 (81.8)
Decrease in total protein	16 (72.7)
Decrease in hemoglobin	20 (90.9)
Increase in total bilirubin	2 (9.1)
Increase in C-reactive protein	20 (90.9)
Increase in white blood cells	11 (50.0)
Increase in creatinine	1 (4.5)
Pain in the implanted limb	18 (81.8)
Gastrointestinal disorders	7 (31.8)
Fever	5 (22.7)
Limb swelling	4 (18.2)
Anemia requiring blood transplantation	2 (9.1)
Hypertension	3 (13.6)
Tachycardia	2 (9.1)
Hyperglycemia	1 (4.5)
Hypotension	1 (4.5)

Discussion

Previous studies have demonstrated clinically effective outcomes of BM-MNC implantation for no-option CLTI patients, particularly those with TAO.^12,15–20 We performed a novel prospective interventional trial to further investigate the efficacy and safety of BM-MNC implantation for patients with TAO. Patients with TAO showing poor improvement despite conventional standard treatment (SPP <30 mmHg) were included in the trial. This trial demonstrated that the SPP, TcPO₂, rest pain scale, and vascular volume of the affected target limb improved significantly by Day 180, and that several other secondary endpoints tended to improve. We speculated that peripheral angiogenesis following BM-MNC implantation led to the development of collateral blood vessels and improved both arterial vascular volume below the knee and peripheral perfusion. Subgroup analyses showed that SPP tended to improve after BM-MNC implantation regardless of baseline SPP and Fontaine classification, particularly in the group with baseline SPP ≥20 mmHg or baseline Fontaine Class III and Class IV. It is possible that the improvement in SPP at Day 180 was not significant in the group with SPP <20 mmHg at baseline because of the small number of participants. Matoba et al reported that the major amputation-free probability at 3 years was 91% in 41 patients,¹⁶ and Idei et al reported that the major amputation-free probability at 4 years was 95% in 26 patients.¹⁷ In the present trial, the major amputation-free probability at 3 years was 95.5%, which is comparable to previous studies. In addition, there were no serious adverse events related to BM-MNC implantation during the present trial. According to these results, we suggest that BM-MNC implantation may be effective for patients with CLTI caused by TAO.

Ohta et al reported on the long-term results of 110 patients with TAO over a 23-year period; in that study, 42.7% of patients underwent major or minor amputation, and major amputation was performed in 13 (11.8%) patients.⁹ Of the 31 patients (46 procedures) who underwent bypass surgery, the respective primary and secondary graft patency rates at 1, 5, and 10 years after surgery were: 41% and 54%; 32% and 47%; and 30% and 39%.⁹ Limb salvage probabilities were 91.4%, 88.6%, and 85.4% at 1, 5, and 10 years after surgery, respectively, and survival probabilities after the initial visit were 97%, 94.4%, and 92.4% at 5, 10, and 20 years, respectively.⁹ In a 5-year follow-up study of 44 patients with TAO undergoing EVT, the respective overall survival and major amputation-free probabilities at 1, 3, and 5 years were: 86.9% and 90.2%; 83.3% and 86.7%; and 83.3% and 86.7%.²¹ In addition, 27.0%, 30.0%, and 41.8% of patients required retreatment after the first EVT at 1, 3, and 5 years, respectively.²¹ In the present study, the respective major amputation-free and overall survival probabilities at 1 and 3 years among 22 patients with TAO were: 95.5% and 95.5%; and 95.5% and 89.5%. The present trial enrolled patients with no-option CLTI who were refractory to conventional standard treatments, including EVT or bypass surgery; nonetheless, the clinical outcomes of BM-MNC implantation were comparable to those of EVT or bypass surgery. In addition, bypass surgery and EVT often required retreatment. However, BM-MNC implantation significantly increased the SPP of the affected target limb after 3 months, which remained high after 6 months. Further, the long-term major amputation-free probability remained unchanged after BM-MNC implantation. Therefore, we believe that BM-MNC implantation may be as effective as conventional standard treatments, such as bypass surgery or EVT, for CLTI patients with TAO.

Tateishi-Yuyama et al reported that TcPO₂ improved significantly 24 weeks after BM-MNC implantation compared with baseline in patients with arteriosclerosis obliterans (ASO; 28 vs. 45 mmHg; P<0.0001).¹² Idei et al reported a significant improvement in TcPO₂ from 1 month after BM-MNC implantation in patients with TAO and that TcPO₂ remained high over a 3-year follow-up.¹⁷ In contrast, TcPO₂ increased significantly 1 month after BM-MNC implantation in patients with ASO, but gradually decreased and returned to baseline values over the follow-up period.¹⁷ In addition, the rest pain scale showed similar improvement from 1 month after BM-MNC implantation in patients with TAO that was maintained,¹⁷ but in patients with ASO there was a gradual increase in the rest pain scale, with values returning to baseline over the 3-year follow-up.¹⁷ Similarly, in the present trial, there were significant improvements in SPP and TcPO₂ by 6 months after BM-MNC implantation. Further, the long-term amputation-free probability was maintained. Therefore, patients with TAO show greater clinical improvement with BM-MNC implantation than do patients with ASO, and this improvement is sustained in the long term. We think that this finding can be attributed to the fact that patients with TAO are younger than those with ASO, have preserved mobility and muscle strength, and are less likely to develop a worsening of vascular lesions due to a lower risk of atherosclerosis. Other factors contributing to the greater clinical improvement in patients with TAO include the improvement in pain at rest caused by angiogenesis following BM-MNC implantation, a reduction in the diameter of ischemic ulceration, and an avoidance of amputation, which may allow patients to exercise more. Exercise stimulates the production of bone marrow-derived endothelial progenitor cells, which are useful for angiogenesis, through a nitric oxide-dependent effect.²² Moreover, exercise improves the skeletal muscle mass and reduces cardiovascular events.^23,24 So, we believe that the improvement in ischemic symptoms following BM-MNC implantation is likely to create a virtuous circle with regard to long-term outcomes in patients with TAO.

For patients with TAO, smoking cessation is most recommended as standard care. Smoking cessation is an absolutely necessary treatment for patients with TAO because smoking worsens peripheral blood flow and exacerbates ischemic symptoms.² However, the ability to stop smoking is a common problem in clinical practice, because it is strongly affected by self-judgment and compliance. In the present trial, the complete success rate of smoking cessation at enrollment was 57.1%. Healthcare providers need to strongly recommend smoking cessation alongside various treatments for CLTI patients with TAO, and we should continue to encourage smoking cessation after BM-MNC implantation.

Together, the results indicate that BM-MNC implantation improved the clinical findings and maintained the long-term amputation-free probability in no-option CLTI patients with TAO. Considering the medical costs associated with major amputation and the social losses incurred by the inability to work because of various symptoms associated with lower limb ischemia, if BM-MNC implantation becomes standard treatment it not only may improve quality of life, but could also have a considerable socially significant effect. BM-MNC implantation will likely become a feasible and safe treatment option for patients with TAO.

Study Limitations

This trial had several limitations. The first limitation is the non-randomized and non-controlled design of the trial. All patients underwent BM-MNC implantation. Therefore, it was difficult to compare the exact efficacy of conventional standard treatments with that of BM-MNC implantation. However, we selected patients with no-option CLTI who were refractory or not indicated for conventional standard treatments, such as drug therapy, exercise therapy, and revascularization. The results of this trial indicate that BM-MNC implantation is effective and safe for these patients. In the future, randomized and controlled trials are required with conventional standard treatments as the control group to evaluate the exact efficacy of BM-MNC implantation for no-option CLTI patients with TAO. Second, the final decision to indicate revascularization was made by a cardiologist, vascular surgeon, or radiologist at each trial institution or referral source hospital. Although this decision is general in clinical practice, it would be preferable for a third party to determine all patients as no-option CLTI at enrollment. In this trial, 38% and 4.8% of patients with TAO had a history of EVT and surgical bypass, respectively, within 3 years before enrollment. We did not collect treatment histories >3 years before enrollment in the CRFs. However, some patients possibly had multiple revascularizations >3 years ago, particularly those previously diagnosed with TAO. To accurately identify all enrolled patients with no-option CLTI, a third party should determine the indication for revascularization at enrollment. Third, although smoking cessation was standard care for patients with TAO, only 57.1% of all patients succeeded to completely stop smoking at enrollment in this trial. Smoking cessation was not included in the inclusion criteria for this trial because it is strongly affected by self-judgment and compliance. Fourth, for all analyses in this trial, the reported P values are 1-sided, and the significance level was set at 1-sided 2.5%. The methods for all statistical analyses in this trial were decided with biostatisticians. Fifth, the sample size was relatively small to evaluate the safety of this treatment. We enrolled 22 patients (FAS: 21 patients) from 6 institutions over 4 years. The target sample size was not achieved because TAO is an intractable and rare disease; however, the required sample number was achieved. In the present trial, we observed no serious adverse events or cardiovascular events related to BM-MNC implantation. Finally, the evaluation items were collected until Day 180 after implantation. We continued general outpatient treatment, including wound care, after 180 days. It was not possible to gauge the maintenance of SPP or TcPO₂ values after Day 180 or the healing of the ischemic ulceration area; however, no patient underwent any amputation of the affected target limb during the trial.

Conclusions

In summary, BM-MNC implantation is likely to become a feasible and safe treatment for CLTI patients with TAO.

Acknowledgments

The authors appreciate the help and support of all the members of the committee for this project and members of the following institutes: Department of Cardiovascular Medicine, Shinshu University School of Medicine; Department of Cardiovascular Regeneration and Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University; Division of Cardiovascular Medicine, Department of Internal Medicine, Kurume University School of Medicine; Department of Cardiology, Yokohama City University Hospital; and Department of Cardiovascular Medicine, Nippon Medical School of Medicine.

Sources of Funding

The authors acknowledge financial support from the Osaka Medical Research Foundation for Intractable Diseases (Grant no. 27-2-47).

Disclosures

K.K. is a member of Circulation Journal’s Editorial Team. The authors declare no other conflicts of interest associated with this manuscript.

IRB Information

This trial was approved by the Ministry of Health, Labour and Welfare as Advanced Medicine B after approval by the Certified Committee for Regenerative Medicine in Kyoto Prefectural University of Medicine in 2017 (ID: NA8150008). Furthermore, the trial was registered again in the Japan Registry of Clinical Trials (jRCT) in 2019 with a revision of the Act on the Safety of Regenerative Medicine (ID: jRCTb050190082). It was also registered with the University Hospital Medical Information Network (UMIN) Clinical Trials Registry (ID: UMIN000027383).

Data Availability

The researchers concerned with this trial have taken participants’ personal information and privacy protection into consideration in accordance with relevant regulations and laws. The deidentified participant data of this trial will not be shared.

References

• 1.   Shionoya S. Buerger’s disease: Diagnosis and management. Cardiovasc Surg 1993; 1: 207–214, doi:10.1177/096721099300100302.
• 2.   Watanabe Y, Miyata T, Shigematsu K, Tanemoto K, Nakaoka Y, Harigai M. Current trends in epidemiology and clinical features of thromboangiitis obliterans in Japan. Circ J 2020; 84: 1786–1796, doi:10.1253/circj.CJ-19-1165.
• 3.   Olin JW, Young JR, Graor RA, Ruschhaupt WF, Batholomew JR. The changing clinical spectrum of thromboangiitis obliterans (Buerger’s disease). Circulation 1990; 82(Suppl): IV3–IV8.
• 4.   Shigematsu H, Shigematsu K. Factors affecting the long-term outcome of Buerger’s disease (thromboangiitis obliterans). Int Angiol 1999; 18: 58–64.
• 5.   Sugimoto M, Miyachi H, Morimae H, Kodama A, Narita H, Banno H, et al. The fate of ischemic limbs in patients with Buerger’s disease based on our 30-year experience: Does smoking have a definitive impact on the late loss of limbs? Surg Today 2015; 45: 466–470, doi:10.1007/s00595-014-0904-6.
• 6.   Hida N, Ohta T. Current status of patients with Buerger disease in Japan. Ann Vasc Dis 2013; 6: 617–623, doi:10.3400/avd.oa.13-00012.
• 7.   Norgren L, Hiatt WR, Dormandy JA, Nehler MR, Harris KA, Fowkes FG. Inter-society consensus for the management of peripheral arterial disease. J Vasc Surg 2007; 45(Suppl S): S5–S67, doi:10.1016/j.jvs.2006.12.037.
• 8.   Miyahara T, Suhara M, Nemoto Y, Shirasu T, Haga M, Mochizuki Y, et al. Long-term results of treatment for critical limb ischemia. Ann Vasc Dis 2015; 8: 192–197, doi:10.3400/avd.oa.15-00074.
• 9.   Ohta T, Ishioashi H, Hosaka M, Sugimoto I. Clinical and social consequences of Buerger disease. J Vasc Surg 2004; 39: 176–180, doi:10.1016/j.jvs.2003.08.006.
• 10.   Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997; 275: 964–967, doi:10.1126/science.275.5302.964.
• 11.   Asahara T, Masuda H, Takahashi T, Kalka C, Pastore C, Silver M, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 1999; 85: 221–228, doi:10.1161/01.res.85.3.221.
• 12.   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: 427–435, doi:10.1016/S0140-6736(02)09670-8.
• 13.   Miyamoto M, Yasutake M, Takano H, Takagi H, Takagi G, Mizuno H, et al. Therapeutic angiogenesis by autologous bone marrow cell implantation for refractory chronic peripheral arterial disease using assessment of neovascularization by ^{99 m}Tc-tetrofosmin (TF) perfusion scintigraphy. Cell Transplant 2004; 13: 429–437, doi:10.3727/000000004783983837.
• 14.   Oda M, Kato K, Toba K, Otaki K, Kitajima T, Ikarashi N, et al. Prognostic factors of critical limb ischemia after autologous bone marrow implantation. J Cardiol 2007; 50: 235–242.
• 15.   Saito Y, Sasaki K, Katsuda Y, Murohara T, Takeshita Y, Okazaki T, et al. Effect of autologous bone-marrow cell transplantation on ischemic ulcer in patients with Buerger’s disease. Circ J 2007; 71: 1187–1192, doi:10.1253/circj.71.1187.
• 16.   Matoba S, Tatsumi T, Murohara T, Imaizumi T, Katsuda Y, Ito M, et al. Long-term clinical outcome after intramuscular implantation of bone marrow mononuclear cells (Therapeutic Angiogenesis by Cell Transplantation [TACT] trial) in patients with chronic limb ischemia. Am Heart J 2008; 156: 1010–1018, doi:10.1016/j.ahj.2008.06.025.
• 17.   Idei N, Soga J, Hata T, Fujii Y, Fujimura N, Mikami S, et al. Autologous bone-marrow mononuclear cell implantation reduces long-term major amputation risk in patients with critical limb ischemia: A comparison of atherosclerotic peripheral arterial disease and Buerger disease. Circ Cardiovasc Interv 2011; 4: 15–25, doi:10.1161/CIRCINTERVENTIONS.110.955724.
• 18.   Yanishi K, Kondo K, Hayashida R, Shintani S, Shibata R, Murotani K, et al. Long-term clinical outcomes survey of bone marrow-derived cell therapy in critical limb ischemia in Japan. Circ J 2018; 82: 1168–1178, doi:10.1253/circj.CJ-17-0510.
• 19.   Shoji K, Yanishi K, Yoshimi R, Hamada N, Kondo K, Fujimoto K, et al. Impact of therapeutic angiogenesis using autologous bone marrow-derived mononuclear cells implantation in critical limb ischemia with scleroderma: Subanalysis of the long-term clinical outcomes survey. Circ J 2019; 83: 662–671, doi:10.1253/circj.CJ-18-1044.
• 20.   Yanishi K, Shoji K, Fujioka A, Hori Y, Yukawa A, Matoba S. Impact of therapeutic angiogenesis using autologous bone marrow-derived mononuclear cell implantation in patients with no-option critical limb ischemia. Ann Vasc Dis 2020; 13: 13–22, doi:10.3400/avd.ra.20-00002.
• 21.   Kim DH, Ko YG, Ahn CM, Shin DH, Kim JS, Jang Y, et al. Immediate and late outcomes of endovascular therapy for lower extremity arteries in Buerger disease. J Vasc Surg 2018; 67: 1769–1777, doi:10.1016/j.jvs.2017.09.020.
• 22.   Laufs U, Werner N, Link A, Endres M, Wassmann S, Nickenig G, et al. Physical training increases endothelial progenitor cells, inhibits neointima formation, and enhances angiogenesis. Circulation 2004; 109: 220–226, doi:10.1161/01.CIR.0000109141.48980.37.
• 23.   Gardner AW, Skinner JS, Cantwell BW, Smith LK. Progressive vs single-stage treadmill tests for evaluation of claudication. Med Sci Sports Exerc 1991; 23: 402–408.
• 24.   Kim M, Ito O, Miura M, Kakihana T, Mori N, Kohzuki M. The effect of exercise training on walking ability and health-related quality of life in patients with moderate to severe peripheral arterial disease. Asian J Hum Serv 2014; 6: 47–58.