Local intramuscular transplantation of autologous bone marrow mononuclear cells for critical lower limb ischaemia

Abstract

Background

Peripheral arterial disease is a major health problem, and in about 1% to 2% of patients, the disease progresses to critical limb ischaemia (CLI), also known as critical limb‐threatening ischaemia. In a substantial number of individuals with CLI, no effective treatment options other than amputation are available, with around a quarter of these patients requiring a major amputation during the following year. This is the second update of a review first published in 2011.

Objectives

To evaluate the benefits and harms of local intramuscular transplantation of autologous adult bone marrow mononuclear cells (BMMNCs) as a treatment for CLI.

Search methods

We used standard, extensive Cochrane search methods. The latest search date was 8 November 2021.

Selection criteria

We included all randomised controlled trials (RCTs) of CLI in which participants were randomly allocated to intramuscular administration of autologous adult BMMNCs or control (either no intervention, conventional conservative therapy, or placebo).

Data collection and analysis

We used standard Cochrane methods. Our primary outcomes of interest were all‐cause mortality, pain, and amputation. Our secondary outcomes were angiographic analysis, ankle‐brachial index (ABI), pain‐free walking distance, side effects and complications. We assessed the certainty of the evidence using the GRADE approach.

Main results

We included four RCTs involving a total of 176 participants with a clinical diagnosis of CLI. Participants were randomised to receive either intramuscular cell implantation of BMMNCs or control. The control arms varied between studies, and included conventional therapy, diluted autologous peripheral blood, and saline. There was no clear evidence of an effect on mortality related to the administration of BMMNCs compared to control (risk ratio (RR) 1.00, 95% confidence interval (CI) 0.15 to 6.63; 3 studies, 123 participants; very low‐certainty evidence). All trials assessed changes in pain severity, but the trials used different forms of pain assessment tools, so we were unable to pool data. Three studies individually reported that no differences in pain reduction were observed between the BMMNC and control groups. One study reported that reduction in rest pain was greater in the BMMNC group compared to the control group (very low‐certainty evidence). All four trials reported the rate of amputation at the end of the study period. We are uncertain if amputations were reduced in the BMMNC group compared to the control group, as a possible small effect (RR 0.52, 95% CI 0.27 to 0.99; 4 studies, 176 participants; very low‐certainty evidence) was lost after undertaking sensitivity analysis (RR 0.52, 95% CI 0.19 to 1.39; 2 studies, 89 participants). None of the included studies reported any angiographic analysis. Ankle‐brachial index was reported differently by each study, so we were not able to pool the data. Three studies reported no changes between groups, and one study reported greater improvement in ABI (as haemodynamic improvement) in the BMMNC group compared to the control group (very low‐certainty evidence). One study reported pain‐free walking distance, finding no clear difference between BMMNC and control groups (low‐certainty evidence). We pooled the data for side effects reported during the follow‐up, and this did not show any clear difference between BMMNC and control groups (RR 2.13, 95% CI 0.50 to 8.97; 4 studies, 176 participants; very low‐certainty evidence). We downgraded the certainty of the evidence due to the concerns about risk of bias, imprecision, and inconsistency.

Authors' conclusions

We identified a small number of studies that met our inclusion criteria, and these differed in the controls they used and how they measured important outcomes. Limited data from these trials provide very low‐ to low‐certainty evidence, and we are unable to draw conclusions to support the use of local intramuscular transplantation of BMMNC for improving clinical outcomes in people with CLI. Evidence from larger RCTs is needed in order to provide adequate statistical power to assess the role of this procedure.

Plain language summary

Does treating reduced blood flow to the legs using cells taken from the bone marrow help improve symptoms?

Why is this question important?

Critical limb ischaemia, or critical limb‐threatening ischaemia, occurs when blood flow to the legs is reduced because of the worsening of peripheral arterial disease. Initially, patients experience cramping leg pain that limits walking (known as intermittent claudication), but over time some patients will experience more severe symptoms including pain at rest, leg ulceration, and gangrene. The available treatment options are very limited when the disease reaches this stage, especially when surgical or catheter revascularisation (a procedure to restore blood flow in blocked arteries or veins) is not an option. Many of these patients will go on to have the affected limb amputated. The use of mononuclear cell therapy (using the patient’s own cells) offers the possibility of an alternative treatment for patients, by supplying cells that could stimulate the formation of stable capillary vessels to improve the blood flow in the affected limb. These cells can be obtained from the bone marrow. They are purified in a laboratory and injected into the large muscle at the back of the lower leg.

What did we find?

We searched for randomised controlled trials (a type of study where participants are randomly assigned to one of two or more treatment groups) that compared treating people using selected bone marrow cells with control (either no intervention, conventional conservative therapy, or placebo (dummy treatment)). We found four studies with a combined total of 176 participants testing the safety and effectiveness of this treatment. The studies compared the cell therapy to different controls. Our analysis showed there is no clear effect of cell therapy on death from any cause. The studies measured pain in different ways, and not all information was reported, so we were unable to pool the data. Individually, three studies did not find any difference in the reduction of pain between the therapy and control groups. One study reported that pain was reduced more in the cell therapy group than in the control group. Pooling data from all four studies showed that cell therapy might reduce amputations, but we are not certain about this because the possible benefit was lost when we redid the analysis excluding data from studies we had concerns about. Three studies reported no improvements in the ankle‐brachial index (a way to measure the blood flow in the leg), whilst one study reported a greater improvement in the ankle‐brachial index in the cell therapy group compared to the control group. No improvements were seen in pain‐free walking distance between groups. No clear difference was seen in side effects between groups.

How confident are we in the evidence?

Our confidence in the evidence was very low because of concerns about how the studies were carried out. The four included studies differed from each other in how they measured effects at different time points; they used different controls; there were small numbers of events and participants overall; and there were differences in individual study results.

Summary of findings

Summary of findings 1. Intramuscular injection of bone marrow mononuclear cells (BMMNC) compared to control for treatment of critical lower limb ischaemia (CLI).

Intramuscular injection of bone marrow mononuclear cells (BMMNC) compared to control for treatment of critical lower limb ischaemia (CLI)
Patient or population: people with CLI who were not suitable for revascularisation and showed no improvement in response to the best standard therapy Setting: hospital Intervention: BMMNCa Comparison: controlb
Outcomes	Anticipated absolute effects (95% CI)*		Relative effect (95% CI)	№ of participants (studies)	Certainty of the evidence (GRADE)	Comments
	Risk with control	Risk with BMMNC
All‐cause mortality (follow‐up: 6 or 12 months)	Study population		RR 1.00 (0.15 to 6.63)	123 (3 RCTs)	⊕⊝⊝⊝ VERY LOWc	No deaths were reported for 2 studies (Barc 2006; Pignon 2017). Li 2013 reported 4 deaths (2 in each group) during 6 months follow‐up; these deaths were not considered to be related to treatment. Information regarding mortality was not available in 1 study (Lindeman 2018).
	32 per 1000	32 per 1000 (5 to 210)
Reduction in pain Various scales including VAS, PISQ; scales ranged from 0 to 4/10/100, where 0 = no pain (follow‐up: 3 to 12 months)	All studies reported on pain, but due to different measurements and incomplete information we were unable to pool the data. See comment			176 (4 RCTs)	⊕⊝⊝⊝ VERY LOWd	Barc 2006 assessed pain with VAS, where 0 was no pain at all and 10 was the most severe pain experienced. Pain levels decreased in both BMMNC and control groups. Li 2013 assessed pain with VAS, but reported the improvement of pain defined as a > 50% decrease in VAS during study follow‐up. Study authors reported a greater reduction in pain in the BMMNC group (P = 0.045). Lindeman 2018 used the PISQ, and reported that no differences were observed in average pain reduction between BMMNC and control group (P = 0.23). Pignon 2017 assessed pain with VAS, where 0 was no pain at all and 100 was the most severe pain experienced. Study authors reported that no differences in pain reduction were observed between the BMMNC and control group.
Incidence of amputation (follow‐up: 6 or 12 months)	Study population		RR 0.52 (0.27 to 0.99)	176 (4 RCTs)	⊕⊝⊝⊝ VERY LOWe	All studies reported the incidence of amputation. A possible small benefit was no longer seen after removal of 2 studies at high risk of bias in sensitivity analysis (RR 0.52, 95% CI 0.19 to 1.39; 89 participants, 2 studies) (Barc 2006; Li 2013).
	250 per 1000	130 per 1000 (68 to 248)
Angiographic analysis	See comment			‐	‐	None of the studies reported angiographic analysis.
Increase in ABI An ABI ratio of 0.9 or less indicates PAD. Values between 0.9 and 1.0 are borderline, and above 1.0 is considered normal (follow‐up: range 1 month to 12 months)	All studies measured ABI, but due to incomplete information we were unable to pool the data. See comment			176 (4 RCTs)	⊕⊝⊝⊝ VERY LOWf	Barc 2006 reported that ABI did not change from baseline during study follow‐up in both BMMNC and control groups. Li 2013 used an absolute increase of > 15% ABI to quantify haemodynamic improvement. Study authors reported that this was greater in the BMMNC group compared to control (P = 0.002). Lindeman 2018 reported no difference in mean ABI between the BMMNC and control groups after 12 months follow‐up (P = 0.50). Pignon 2017 did not show any changes in median ABI value between groups.
Increase in PFWD (m) (follow‐up: 12 months)	See comment			53 (1 RCT)	⊕⊕⊝⊝ LOWg	Only Lindeman 2018 reported PFWD, finding no clear difference between groups. The mean PFWD for the treatment and control groups at 12 months follow‐up was 128 ± 71 m vs 160 ± 11 m, P = 0.87.
Side effects and complications (follow‐up: 6 or 12 months)	Study population		RR 2.13 (0.50 to 8.97)	176 (4 RCTs)	⊕⊝⊝⊝ VERY LOWh	All trials reported adverse events. 2 studies reported that no identifiable treatment‐related adverse events were observed, and that the therapy was well‐tolerated (Barc 2006; Pignon 2017). Li 2013 reported adverse events in both BMMNC and control groups (3 vs 1 fever, 1 vs 0 MI, 0 vs 1 stroke). There were no differences in the incidence of adverse events between groups, and the therapy was well‐tolerated Lindeman 2018 reported that leukaemia occurred in 1 participant during the follow‐up period. It is unclear if this was related to the procedure. After applying the sensitivity analysis by removing 2 studies at high risk of bias (Barc 2006; Li 2013), the significance of the results did not change (RR 2.69, 95% CI 0.11 to 63.18).
	23 per 1000	48 per 1000 (11 to 204)
*The risk in the intervention group (and its 95% confidence interval) is based on the assumed risk in the comparison group and the relative effect of the intervention (and its 95% CI). ABI: ankle‐brachial index;BMMNC: bone marrow mononuclear cells; CI: confidence interval; CLI: critical limb ischaemia;MI: myocardial infarction; PAD: peripheral arterial disease; PFWD: pain‐free walking distance;PISQ: pain inventory score questionnaire; RCT: randomised controlled trial; RR: risk ratio; VAS: visual analogue scale
GRADE Working Group grades of evidence High certainty: we are very confident that the true effect lies close to that of the estimate of the effect. Moderate certainty: we are moderately confident in the effect estimate: the true effect is likely to be close to the estimate of the effect, but there is a possibility that it is substantially different. Low certainty: our confidence in the effect estimate is limited: the true effect may be substantially different from the estimate of the effect. Very low certainty: we have very little confidence in the effect estimate: the true effect is likely to be substantially different from the estimate of effect.

^aThe stem cells used in the included RCTs originate from bone marrow. All four trials used mononuclear cells collected during the harvesting procedure from bone marrow and implanted into affected limbs (Barc 2006; Li 2013; Lindeman 2018; Pignon 2017).
^bThe control arms varied across all included RCTs: conventional therapy (Barc 2006), 0.9% sodium chloride (saline) (Li 2013), diluted autologous peripheral blood (Lindeman 2018), 30 mL saline with 4 mL autologous peripheral blood (Pignon 2017).
^cWe downgraded a total of three levels due to concerns related to risk of bias, imprecision (few participants and events), and inconsistency (wide CIs and clinical heterogeneity).
^dWe downgraded one level for concerns related to risk of bias, one level for imprecision (few participants and events), and one level for inconsistency (heterogeneity due to different control arms).
^eWe downgraded one level for concerns related to risk of bias, one level for imprecision (few participants and events), and one level for inconsistency (heterogeneity due to different control arms).
^fWe downgraded one level for concerns related to risk of bias, one level for imprecision (few participants and events), and one level for inconsistency (wide CIs, and heterogeneity due to different control arms).
^gWe downgraded one level for imprecision (few participants and events) and one level for inconsistency (wide CIs, heterogeneity due to different control arms).
^hWe downgraded one level for concerns related to risk of bias, one level for imprecision (few participants and events), and one level for inconsistency (wide CIs).

Background

Description of the condition

Peripheral arterial disease is a major health problem with a total disease prevalence of 3% to 10% that increases to 15% to 20% in individuals over the age of 70 years (Hirsch 2006). As the disease progresses, about 1% to 2% of patients develop critical limb ischaemia (CLI), also known as critical limb‐threatening ischaemia, which is characterised by chronic ischaemic rest pain, ischaemic ulcers, or gangrene (Norgren 2007).

The current mainstay treatment of CLI has been surgical or catheter‐based revascularisation. However, for a substantial number of patients revascularisation is not be feasible because of the involvement of distal vessels. For individuals with CLI who are not candidates for revascularisation, around a quarter will require a major amputation during the following year (Norgren 2007).

Description of the intervention

Pre‐clinical studies of the use of mononuclear cells from the bone marrow has shown promising results in animal models with CLI. These data demonstrated an improvement in capillary density in hindlimb ischaemia and promoted collateral vessel formation (Shintani 2001). On the basis of these results in animals, clinical trials were started in order to test cell therapy with autologous bone marrow mononuclear cells (BMMNCs) in people with ischaemic lower limbs.

At present, the procedure is as follows. Mononuclear cells are derived either directly from bone marrow aspiration (Higashi 2004; Tateishi‐Yuyama 2002), or through mobilisation into the peripheral blood using granulocyte colony‐stimulating factor (G‐CSF) (Huang 2004; Huang 2005b). In the first procedure, bone marrow cells are usually collected (mostly under general anaesthesia) from the iliac crest. Thereafter, the BMMNCs are enriched away from other bone marrow cells in sterile conditions in a laboratory. In the second procedure, mononuclear cells mobilised into the peripheral blood following G‐CSF administration for four to five days are collected from a peripheral blood sample and then enriched from other blood cells in sterile conditions. In both procedures, the enriched mononuclear cells are directly implanted into the gastrocnemius muscle of the ischaemic leg.

How the intervention might work

Although the exact mechanism of action of mononuclear cell implantation has not been fully elucidated, BMMNCs have been shown to secrete a number of angiogenic cytokines (Shintani 2001; Takahashi 2006). It therefore seems that implantation into ischaemic limbs could enhance angiogenesis through paracrine mechanisms by supplying endothelial progenitor cells and providing multiple angiogenic factors or cytokines. These combined mechanisms may subsequently lead to the formation of stable capillary vessels and so reverse the ischaemic status of the affected limb.

Why it is important to do this review

Since mononuclear cell implantation has emerged as a novel intervention in clinical practice for CLI, it is important that a systematic review is undertaken in order to assess the safety and efficacy of this intervention. We have therefore updated the systematic review first published in 2014, Moazzami 2014, in order to identify recent evidence regarding the potential therapeutic benefits and possible harms of local intramuscular BMMNC implantation for people with CLI.

Objectives

To evaluate the benefits and harms of local intramuscular transplantation of autologous adult bone marrow mononuclear cells (BMMNCs) as a treatment for critical limb ischaemia (CLI).

Methods

Criteria for considering studies for this review

Types of studies

We included randomised controlled trials (RCTs).

Types of participants

We included participants with a clinical diagnosis of critical limb ischaemia (CLI) who had been admitted to hospital for treatment. We included participants who were not candidates for open or endovascular revascularisation (Rutherford category 5/6) according to angiographic evidence of superficial femoral artery or infrapopliteal disease in the affected limb, as well as those who did not show any evidence of improvement in response to best standard therapy in the previous four weeks. There was no age restriction.

Types of interventions

We included studies involving the administration of autologous adult BMMNCs by direct implantation into the gastrocnemius muscle of ischaemic legs of participants as a treatment for CLI, compared to either no intervention, conventional conservative therapy (e.g. pharmacological treatment of pain, wound care, or bed rest), or administration of an inert placebo such as isotonic saline.

Types of outcome measures

Primary outcomes

• All‐cause mortality

• Reduction in pain, as assessed by analgesic requirements or a pain analogue scale

• Incidence of amputation (minor or major, where major amputation is above the ankle, and minor amputation is below the ankle or part of the foot)

Secondary outcomes

• Angiographic analysis (changes in the number of visible vessels or any change in the intensity or apparent size of previously visible vessels)

• Increase in ankle‐brachial index (ABI)

• Increase in pain‐free walking distance (PFWD)

• Side effects and complications, such as local or systemic inflammation, cardiovascular abnormalities, and thromboembolic complications

Search methods for identification of studies

Electronic searches

The Cochrane Vascular Information Specialist conducted systematic searches of the following databases for RCTs and controlled clinical trials without language, publication year, or publication status restrictions:

• Cochrane Vascular Specialised Register via the Cochrane Register of Studies (CRS Web searched from 17 February 2014 to 8 November 2021);

• Cochrane Central Register of Controlled Trials (CENTRAL); Cochrane Register of Studies Online (CRSO 2021, Issue 10) (searched on 8 November 2021);

• MEDLINE (Ovid MEDLINE Epub Ahead of Print, In‐Process & Other Non‐Indexed Citations, Ovid MEDLINE Daily and Ovid MEDLINE) (searched from 1 January 2017 to 8 November 2021);

• Embase Ovid (searched from 1 January 2017 to 8 November 2021);

• CINAHL EBSCO (Cumulative Index to Nursing and Allied Health Literature) (searched from 1 January 2017 to 8 November 2021);

• AMED Ovid (Allied and Complementary Medicine) (searched from 1 January 2017 to 8 November 2021).

The Information Specialist modelled search strategies for other databases on the search strategy designed for CENTRAL. Where appropriate, search strategies were combined with adaptations of the Highly Sensitive Search Strategy designed by Cochrane for identifying RCTs and controlled clinical trials (as described in Chapter 6 of the Cochrane Handbook for Systematic Reviews of Interventions) (Lefebvre 2021). Search strategies for major databases are provided in Appendix 1.

The Information Specialist searched the following trials registries on 8 November 2021:

• World Health Organization International Clinical Trials Registry Platform (who.int/trialsearch);

• US National Institutes of Health Ongoing Trials Register ClinicalTrials.gov (clinicaltrials.gov).

Searching other resources

We checked the reference lists of papers and reports retrieved from the electronic searches to identify additional potentially relevant studies.

Data collection and analysis

Selection of studies

Three review authors (KM, EF, and ZM) screened the titles and abstracts of references identified by the search, and two review authors (BM and ZM or ZZ) independently assessed their eligibility for inclusion in the review. Any disagreements were resolved by consensus or by a discussion with the third review author (KM). We obtained full versions of articles deemed potentially relevant based on title or abstract, and three review authors (BM, AR, and ED) assessed these independently against the inclusion criteria. We recorded the reasons for exclusion of any study excluded at the full‐text stage in the Characteristics of excluded studies table.

Data extraction and management

Three review authors (BM, AR, and ZM) independently extracted and recorded the dichotomous and continuous data for the prespecified outcomes on forms developed by Cochrane Vascular. We recorded details on study design, setting, participant numbers, inclusion and exclusion criteria, cell type, route of delivery, control used, outcomes, funding and declarations of interest made by the study authors. Any disagreements were resolved by another review author (ED); if necessary, we sought additional information from the study authors.

Assessment of risk of bias in included studies

We used the Cochrane risk of bias 1 (RoB 1) tool as described in the Cochrane Handbook for Systematic Reviews of Interventions to evaluate risk of bias of the included studies (Higgins 2017). We assessed the following risk of bias domains: randomisation and allocation (selection bias), blinding (performance bias and detection bias), incomplete outcome data (attrition bias), selective reporting (reporting bias), and other potential sources of bias, judging each domain to be either low, high, or unclear risk of bias according to the guidance in Higgins 2017.

Measures of treatment effect

We expressed the measure of effect as risk ratios (RRs) with 95% confidence intervals (CI) for each dichotomous outcome. Where continuous scales of measurement were used to assess the effects of treatment, we used mean difference (MDs) with 95% CI. When different scales were used in the different studies, we standardised the results where possible and combined them using standardised mean difference (SMD) with 95% CI.

Unit of analysis issues

The unit of analysis was the individual participant randomised to either BMMNC therapy or a control group.

Dealing with missing data

Where necessary, we sought missing data and data regarding participant demographics and outcome measures by contacting the corresponding study author. We planned that if some outcome data remained missing despite our attempts at retrieval, we would exclude the trials from the analyses where there were more than 10% incomplete or missing entries for each variable.

Assessment of heterogeneity

We explored and assessed heterogeneity using the I² and Q statistics, and by the subjective judgement of the comparability of participants, interventions, and outcomes. We planned to assess statistical heterogeneity of trial data by using the Mantel‐Haenszel Chi² test of heterogeneity and the I² statistic of heterogeneity (Higgins 2021). We considered data to be heterogeneous if P was less than 0.10 for the first method. For the I² method, we used the guidelines on interpretation described in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2021), which suggest that an I² statistic of 0% to 40% might not be important; 30% to 60% may represent moderate heterogeneity; 50% to 90% may represent substantial heterogeneity; and 75% to 100% considerable heterogeneity.

Assessment of reporting biases

We planned that if sufficient trials were available, we would use funnel plots to assess publication bias. However, as only four studies were included in the review, this was not possible.

Data synthesis

We only undertook meta‐analyses where the treatments, participants, and underlying clinical questions were similar enough for pooling to be meaningful. The overall treatment effect was estimated by the pooled RR with 95% CI. Since there were differences in the methods of the studies (sources of error were both within‐study and between‐study variance), we used a random‐effects model to perform the analysis. Each test for significance was two‐tailed.

Subgroup analysis and investigation of heterogeneity

We planned to perform subgroup analyses by the type of disease (atherosclerosis versus thromboangitis obliterans) if sufficient trials were available. Performing subgroup analyses was not feasible owing to inadequate information regarding participants with or without significant comorbidity, gender, ethnicity, and different age groups.

Sensitivity analysis

We planned to undertake sensitivity analyses to examine the robustness of the observed findings in relation to a number of factors including study quality and patient type if sufficient studies were identified. We performed sensitivity analysis for the outcomes amputation and side effects and complications by removing two studies at high risk of bias (Barc 2006; Li 2013). We judged studies to be at high risk of bias if any of the following domains were at high risk of bias or if all of the following domains were at unclear risk of bias: random sequence generation and allocation concealment (selection bias), blinding of participants and personnel (performance bias), and blinding of outcome assessment (detection bias).

Summary of findings and assessment of the certainty of the evidence

We created a summary of findings table to present the main findings of this review using GRADEpro GDT software (GRADEpro GDT), and the guidelines provided in Chapter 14 of the Cochrane Handbook for Systematic Reviews of Interventions (Schünemann 2021). We used the five GRADE criteria (risk of bias, inconsistency, imprecision, indirectness, and publication bias) to assess the certainty of the body of evidence (GRADE 2004). We justified all decisions to downgrade the certainty of the evidence in footnotes. We included the outcomes considered to be of most clinical relevance in Table 1, as follows.

• All‐cause mortality

• Reduction in pain

• Incidence of amputation

• Angiographic analysis

• ABI

• PFWD

• Side effects and complications

Results

Description of studies

Search results are presented in Figure 1.

1.

Study flow diagram.

Results of the search

We included three new studies in this update (Li 2013; Lindeman 2018; Pignon 2017). We excluded one previously included study following clarification of our inclusion criteria (Huang 2005a). In total, we excluded 38 new studies (Benoit 2011; BONMOT 2008; Burt 2010; Dong 2018; Du 2017; Flugelman 2017; Frogel 2017; Horie 2018; Huang 2005a; Iafrati 2016; JUVENTAS 2008; Korymasov 2009; Majumdar 2015; Molavi 2016; Murphy 2017; NCT01584986; NCT02336646; NCT03174522; NCT03214887; NCT03304821; NCT03339973; Niven 2017; Ohtake 2017; Perin 2017; Poole 2013; Prochazka 2010; PROVASA 2011; RESTORE‐CLI 2012; Sharma 2021; Skóra 2015; Teraa 2015; Tournois 2015; Wang 2014; Wang 2017; Wang 2018; Wijnand 2018; Zhou 2017a; Zhou 2017b). We identified three new ongoing studies (NCT00753025; NCT01446055; NCT02454231). See Characteristics of included studies, Characteristics of excluded studies, and Characteristics of ongoing studies.

Included studies

For details, see Characteristics of included studies.

We identified four included studies involving a total of 176 participants for this review (Barc 2006; Li 2013; Lindeman 2018; Pignon 2017). All studies were RCTs comparing the effect of intramuscular injections of BMMNCs versus control. Barc 2006 compared the effect of intramuscular injections of BMMNCs versus control in people with CLI. Control therapy was described by the authors as standard conservative therapy, which was also given to the treatment group. Li 2013 investigated the effect of administration of BMMNCs in comparison with 0.9% sodium chloride (also known as saline or saline solution). Lindeman 2018 compared administration of BMMNCs to placebo (diluted autologous peripheral blood). In Pignon 2017, participants who received BMMNCs were compared with participants who received placebo (30 mL saline with 4 mL autologous peripheral blood). One trial was a multicentre study conducted in seven academic centres in France (Pignon 2017); the other three studies were single centre (Barc 2006; Li 2013; Lindeman 2018).

Trial duration

The duration of follow‐up varied from six to 12 months. The treatment duration in two studies lasted six months (Barc 2006; Li 2013), and in two studies participants were followed up for 12 months (Lindeman 2018; Pignon 2017). All studies used the end of the treatment as the final follow‐up time for the treatment phase.

Sample sizes

All of the included studies had small sample sizes, ranging from 29 participants in Barc 2006 to 58 participants in Li 2013. Overall, 176 participants were included. Of these, 88 participants were randomised to receive BMMNCs, and 88 participants were randomised to control. In Barc 2006, 14 participants were randomised to the BMMNC treatment group and 15 to the control group (conventional therapy). Li 2013 randomised 58 participants to BMMNC and 29 to control (saline). In Lindeman 2018, 53 participants were randomised, 28 to BMMNCs and 25 to placebo (diluted autologous peripheral blood). In Pignon 2017, 17 participants received BMMNCs and 19 participants received placebo (30 mL saline with 4 mL autologous peripheral blood).

Participants

All included studies involved participants with a diagnosis of CLI. The most common aetiology of CLI amongst the included studies was atherosclerosis obliterans. Data regarding participant age, sex, the severity of limb ischaemia, and baseline comorbidities (diabetes mellitus, hypertension, and chronic obstructive pulmonary disease (COPD)) were assessed and compared between groups by the individual studies. Barc 2006 included participants with CLI at risk of amputation who showed no progress after eight weeks of conventional therapy and with no option for operative therapy. Li 2013 included participants with CLI without improvement after a minimum of four weeks on conventional therapy. Lindeman 2018 included participants with end‐stage peripheral arterial disease and CLI without improvement after six months of optimal therapy and with no revascularisation options. Pignon 2017 included participants with CLI and no improvement after medical therapy after a duration of 12 months.

Characteristics of bone marrow cell therapy

The autologous mononuclear cells were obtained from bone marrow in all of the included studies (Barc 2006; Li 2013; Lindeman 2018; Pignon 2017). In Barc 2006, the isolation of mononuclear cells from collected marrow was performed with Baxter Fenwal CS 3000 plus blood cell separator. In Li 2013, BMMNCs were isolated from the bone marrow by density gradient centrifugation with lymphocyte separating fluid. In Lindeman 2018, the collected bone marrow suspension was filtered and concentrated to a final volume of 40 mL on the COBE Spectra Apheresis System (Gambro, Stockholm, Sweden) without further manipulation. In Pignon 2017, two different approaches were performed for BMMNC separation across centres, including blood‐cell separator using COBE Spectra, version 4, Bone Marrow Processing Program (Gambro BCT, Lakewood, CO, USA), and blood‐cell separator requiring a Ficoll density‐gradient for isolation of the BMMNC (COBE 2991, Gambro BCT).

The median cell dosage of mononuclear cell count varied from 1.3 x 10⁹, in Pignon 2017, to 1.7 x 10⁹, inLindeman 2018. The median number of CD34(+) cells were 1 x 10⁷, 33.5 x 10⁶, and 48.8 x 10⁶ in Li 2013, Pignon 2017, and Lindeman 2018, respectively. In Barc 2006, the exact number of mononuclear cells was not stated. There was no standard definition for high versus low cell amounts across all studies. Intramuscular cell implantation was the route of administration in all studies. The standard procedure for cell implantation was performed following multiple intramuscular injections into the gastrocnemius muscle. The cells were administered multiple times, ranging from four times in Barc 2006 to 40 times in Lindeman 2018.

None of the included studies provided additional information regarding the teams responsible for the administration of the BMMNC or deciding on the indication of open or endovascular revascularisation, or details of the angiographic assessments.

Outcomes

The outcomes reported in the individual trials are reported in the Characteristics of included studies tables and include:

• all‐cause mortality (Barc 2006; Li 2013; Pignon 2017);

• increase in ankle‐brachial index (ABI) (Barc 2006; Lindeman 2018; Pignon 2017);

• reduction in pain assessed by analgesic requirements or a pain analogue scale (Barc 2006; Li 2013; Lindeman 2018; Pignon 2017);

• progression of disease in terms of the incidence of amputation (Barc 2006; Li 2013; Lindeman 2018; Pignon 2017);

• increase in pain‐free walking distance (PFWD) (Lindeman 2018);

• measurement of transcutaneous pressure of oxygen (TcPO₂) (Pignon 2017);

• side effects (Barc 2006; Li 2013; Lindeman 2018; Pignon 2017).

Excluded studies

We excluded 38 additional studies in this update (Benoit 2011; BONMOT 2008; Burt 2010; Dong 2018; Du 2017; Flugelman 2017; Frogel 2017; Horie 2018; Huang 2005a; Iafrati 2016; JUVENTAS 2008; Korymasov 2009; Majumdar 2015; Molavi 2016; Murphy 2017; NCT01584986; NCT02336646; NCT03174522; NCT03214887; NCT03304821; NCT03339973; Niven 2017; Ohtake 2017; Perin 2017; Poole 2013; Prochazka 2010; PROVASA 2011; RESTORE‐CLI 2012; Sharma 2021; Skóra 2015; Teraa 2015; Tournois 2015; Wang 2014; Wang 2017; Wang 2018; Wijnand 2018; Zhou 2017a; Zhou 2017b), for a total of 73 excluded studies (see Characteristics of excluded studies).

The main reasons for exclusion were as follows.

• Fifty studies investigated the effect of preparations of cells other than mononuclear cells. These included bone marrow cells (NCT00498069), CD34+ cells (Dong 2013; Dong 2018; Madaric 2011; NCT00616980), peripheral blood‐derived stem cell therapy (Capiod 2009; Huang 2005a; NCT00922389; Niven 2017; Ohtake 2017; Szabo 2013), CD133+ cells (Burt 2010; Korymasov 2009; NCT00913900), bone marrow concentrate (BONMOT 2008; Gurunathan 2009; Iafrati 2011; Iafrati 2016; Murphy 2017; NCT00595257; NCT01049919; NCT01245335; Prochazka 2010; Wang 2017), allogeneic mesenchymal stem cells (MSCs) (Du 2017; Gupta 2013; Lu 2011; Majumdar 2015; Mohamed 2020; NCT02336646; Pawan 2012; Wang 2018; Wijnand 2018), bone marrow mesenchymal stem cells (BMMSCs) (Chen 2009; Debin 2008; NCT00955669), adult stem cells (Lasala 2011), bone marrow‐derived aldehyde dehydrogenase bright cells (Perin 2011), expanded autologous bone marrow‐derived tissue, Ixmyelocel‐T and vascular repair cells (RESTORE‐CLI 2012), autologous angiogenic cell precursors (NCT01584986), Rexmyelocel‐T (REX‐001) (NCT03174522), granulocyte‐macrophage colony stimulating factor (GM‐CSF) (NCT03304821; Poole 2013; Zafarghandi 2010), allogeneic ABCB5‐positive stem cells (NCT03339973), venous endothelial cells (ECs) (Flugelman 2017), aldehyde dehydrogenase (ALDH) bright cells (Perin 2017), vascular endothelial growth factor (VEGF) 165 gene‐modified PB CD34+ cells (Zhou 2017b), circulating blood‐derived progenitor cells (Frogel 2017), and autologous cell therapy products (Tournois 2015).

• Ten studies did not include people with CLI (Holzinger 1994; Horie 2018; Kirana 2007; Kirana 2012; NCT00306085; NCT00539266; NCT03214887; Subramaniyam 2009; Zhang 2010; Zhou 2017a).

• In six studies, cells were not administered intramuscularly, and the intra‐arterial route of administration was applied (JUVENTAS 2008; NCT00282646; PROVASA 2011; Sharma 2021; Teraa 2015; Walter 2011).

• In one study, the active intervention arm consisted of BMMNCs co‐administered with peripheral blood mononuclear cells (PBMNCs) (Zhao 2008).

• In one study, BMMNCs were co‐administered with a selective 5‐HT(2A) antagonist in the treatment arm (Higashi 2010).

• In one study, bone marrow was processed into 40 mL of concentrate (Benoit 2011).

• In one study, non‐cell‐based products were compared to cell treatment (Wang 2014).

• In one study, the therapeutic effects of intramuscular and intra‐arterial delivery of bone marrow cells were compared (Klepanec 2012).

• One study evaluated the safety and efficacy of repeated bone marrow mononuclear cell injections compared with a single bone marrow mononuclear cell injection (Molavi 2016).

• One study used BMMNCs in combination with gene therapy (Skóra 2015).

Ongoing studies

We identified three new ongoing studies (NCT00753025; NCT01446055; NCT02454231). For details, see Characteristics of ongoing studies.

Risk of bias in included studies

Please refer to Figure 2 and Figure 3. Details and justification for the risk of bias ratings for each included study are presented in the risk of bias tables in Characteristics of included studies.

2.

Risk of bias graph: review authors' judgements about each risk of bias item presented as percentages across all included studies.

3.

Risk of bias summary: review authors' judgements about each risk of bias item for each included study.

Allocation

All included trials explicitly stated that randomisation occurred; however, in two studies the method used to generate the random sequence was not described, and we judged them to be at unclear risk of selection bias (Barc 2006; Li 2013). The randomisation methods and allocation were adequately performed in two trials, and these were judged as being at low risk of selection bias (Lindeman 2018; Pignon 2017).

Blinding

In two trials, both outcome assessors and participants were blinded, and these were judged as being at low risk of bias (Lindeman 2018; Pignon 2017). One study was single‐blinded, and was thus judged as being at unclear risk of performance and detection bias (Li 2013). One study provided insufficient information concerning blinding and was judged to be at unclear risk of performance and detection bias (Barc 2006).

Incomplete outcome data

We assessed three studies as having a low risk of attrition bias (Barc 2006; Lindeman 2018; Pignon 2017). We assessed the remaining study as having a high risk of attrition bias, as substantial numbers of participants were not evaluated for some outcomes (Li 2013).

Selective reporting

We did not identify any reporting bias in two studies (Li 2013; Lindeman 2018). We judged two studies as being at high risk of selection bias, as ABI and pain were not reported in sufficient detail to use in the analysis (Barc 2006; Pignon 2017).

Other potential sources of bias

We identified no other potential sources of bias in the four included studies (Barc 2006; Li 2013; Lindeman 2018; Pignon 2017).

Effects of interventions

See: Table 1

See Table 1.

All‐cause mortality

No deaths were reported during the study period in two included trials (Barc 2006; Pignon 2017). Information regarding mortality was not available in one study (Lindeman 2018). One study reported four deaths during study follow‐up (two in the therapy group and two in the control group); the study authors stated that none of the deaths were related to treatment (Li 2013). Pooled data of three studies showed no clear effect of BMMNC therapy on all‐cause mortality (risk ratio (RR) 1.00, 95% confidence interval (CI) 0.15 to 6.63; 3 studies, 123 participants; P = 1.0; very low‐certainty evidence; Analysis 1.1). We downgraded the certainty of the evidence a total of three levels due to risk of bias (two studies with unclear risk of selection, performance, and detection bias); imprecision (few participants and events); and inconsistency (wide CIs and clinical heterogeneity) (Table 1).

1.1. Analysis.

Comparison 1: BMMNC versus control, Outcome 1: All‐cause mortality

Reduction in pain, as assessed by analgesic requirements or a pain analogue scale

We were unable to pool data, as different measures were used to assess pain, so we have provided a narrative report.

In Barc 2006, ischaemic pain was assessed with a visual analogue pain scale (VAS) with 10 levels, where 0 was no pain at all and 10 was the most severe pain experienced. The study reported that pain decreased in both groups. Data were not reported in sufficient detail to use.

Li 2013 used the same assessment method (VAS), but reported improvement of pain defined as a > 50% decrease in pain scores during study follow‐up. Study authors reported a significant difference for pain reduction between the BMMNC and control groups (42% versus 12%, P = 0.045).

Lindeman 2018 assessed both the severity of pain and the impact of pain on daily activities by a Pain Inventory Score Questionnaire. The mean pain score reduction at 12 months in the BMMNC group was 4.6 ± 2.6, compared to 4.8 ± 1.9 in the control group (P = 0.23). Overall, no differences were observed in average pain reduction between the BMMNC group and the control group (diluted autologous peripheral blood).

Pignon 2017 evaluated pain severity using a VAS scoring system ranging from 0 to 100. Study authors reported that no differences in pain reduction were observed between participants who received BMMNC and those who received control (diluted autologous peripheral blood), but data were not reported in sufficient detail to include in the analysis.

Overall, we downgraded the certainty of the evidence for pain to very low due to risk of bias, imprecision (few participants and events), and inconsistency (clinical heterogeneity) (Table 1).

Incidence of amputation

All four studies examined the rate of amputations during their study period (Barc 2006; Li 2013; Lindeman 2018; Pignon 2017). The pooled findings showed that compared with control (including conventional therapy, diluted autologous peripheral blood, and saline), intramuscular mononuclear cell implantation was possibly associated with a small reduction in risk of amputation (RR 0.52, 95% CI 0.27 to 0.99; 4 studies, 176 participants; P = 0.05; very low‐certainty evidence; Analysis 1.2). This possible slight benefit was lost after applying sensitivity analysis by removing two studies at a high risk of bias (RR 0.52, 95% CI 0.19 to 1.39; 2 studies, 89 participants; P = 0.19) (Analysis 1.3) (Barc 2006; Li 2013). We downgraded the certainty of the evidence due to risk of bias (two studies with unclear risk of selection, performance, and detection bias); imprecision (few participants and events); and inconsistency (wide CIs) (Table 1).

1.2. Analysis.

Comparison 1: BMMNC versus control, Outcome 2: Amputation

1.3. Analysis.

Comparison 1: BMMNC versus control, Outcome 3: Amputation ‐ sensitivity analysis

Angiographic analysis

None of the included studies reported data on angiographic assessments.

Increase in ankle‐brachial index (ABI)

All four included studies measured ABI, but incomplete information precluded the pooling of data, therefore we have provided a narrative report. Barc 2006 reported that ABI did not change from baseline during study follow‐up in either the BMMNC or the control group. Li 2013 used an absolute increase of > 15% ABI to quantify haemodynamic improvement, and reported that this was greater in the therapy group compared to the control group (52% versus 5%, P = 0.002). Lindeman 2018 reported no difference in mean ABI between BMNC treatment and control groups after 12 months follow‐up (mean ABI: 0.68 ± 0.32 versus 0.50 ± 2.0, respectively; P = 0.50). Pignon 2017 reported the median ABI in a figure which did not show any changes in ABI value between groups; we were unable to obtain the specific data. We downgraded the certainty of the evidence to very low due to concerns related to risk of bias, imprecision (few participants and events), and inconsistency (clinical heterogeneity) (Table 1).

Increase in pain‐free walking distance (PFWD)

Only one study evaluated the effect of intramuscular BMMNC implantation on PFWD (Lindeman 2018). There was no clear difference in mean PFWD between participants who received BMMNC and those who received control (diluted autologous peripheral blood). The mean (± standard deviation) PFWD for the treatment and control groups at 12 months follow‐up was 128 ± 71 m compared to 160 ± 11 m, respectively; P = 0.87. We downgraded the certainty of the evidence to low due to imprecision (few participants and events) and inconsistency (clinical heterogeneity) (Table 1).

Side effects and complications

All of the included trials reported adverse events. Two studies reported that no identifiable treatment‐related adverse events were observed, and that therapy was well‐tolerated (Barc 2006; Pignon 2017). Lindeman 2018 reported that leukaemia occurred in one participant during the follow‐up period. However, no further information was provided to permit an assessment of whether this adverse event was thought to be directly related to the procedure. Whilst Li 2013 reported some adverse events in both the BMMNC and the control group (3 versus 1 fever, 1 versus 0 myocardial infarction, 0 versus 1 stroke, respectively), they reported there were no significant differences in the incidence of adverse events between groups and that therapy was well‐tolerated. The pooled data showed no clear difference between groups in the incidence of side effects and complications (RR 2.13, 95% CI 0.50 to 8.97; 4 studies, 176 participants; P = 0.30; Analysis 1.4). The overall effect was unchanged after sensitivity analysis in which two studies at high risk of bias were removed (RR 2.69, 95% CI 0.11 to 63.18; 2 studies, 89 participants; P = 0.54; Analysis 1.5) (Barc 2006; Li 2013). We downgraded the certainty of the evidence to very low due to risk of bias, imprecision (few participants and events), and inconsistency (wide CIs and clinical heterogeneity) (Table 1).

1.4. Analysis.

Comparison 1: BMMNC versus control, Outcome 4: Side effects and complications

1.5. Analysis.

Comparison 1: BMMNC versus control, Outcome 5: Side effects and complications ‐ sensitivity analysis

Subgroup and sensitivity analysis

Performing subgroup analysis was precluded by inadequate information regarding participants with or without significant comorbidity, gender, ethnicity, and different age groups.

We performed sensitivity analysis for the outcomes amputation (Analysis 1.3) and side effects and complications (Analysis 1.5) by removing the two studies at high risk of bias (Barc 2006; Li 2013). Results are reported above.

Discussion

Summary of main results

See Table 1.

A large body of evidence exists regarding the use of bone marrow mononuclear cells (BMMNCs) for the treatment of patients with various haematological malignancies, but their role in the treatment of other diseases, including critical limb ischaemia (CLI), has not been exclusively addressed. Moreover, the findings of this review suggest that there is limited evidence to support this practice at present. In the current update, we included four studies with a total of 176 participants who were randomised to receive either intramuscular BMMNC implantation or control for the treatment of CLI. There was heterogeneity between study control arms, with groups receiving either conventional therapy, diluted autologous peripheral blood, or 0.9% sodium chloride (saline) solution.

There were no mortality events related to the administration of BMMNCs, and analysis did not show any clear difference between the BMMNC and control groups with respect to mortality risk. We deemed the certainty of this evidence to be very low due to risk of bias, imprecision, and inconsistency. All trials assessed changes in pain severity with different forms of pain assessment tools, and so we were unable to pool data. Three studies individually reported that no differences in pain reduction were observed between participants who received BMMNC and those who received control. Li 2013 reported that reduction in rest pain was greater in the BMMNCs group compared to the control group. We downgraded the certainty of the evidence for this outcome to very low due to risk of bias, imprecision, and inconsistency.

All four trials reported on the rate of amputation at the end of the study period. Analysis showed that BMMNC treatment possibly reduced the risk of amputation slightly compared to control treatment (very low‐certainty evidence). This possible benefit was lost after removal of two studies at high risk of bias in a sensitivity analysis. We downgraded the certainty of the evidence to very low due to risk of bias, imprecision, and inconsistency. The evidence for risk of amputation following BMMNC treatment is uncertain.

None of the included studies reported angiographic analysis.

All of the included studies measured ankle‐brachial index (ABI) index. Incomplete information precluded the pooling of data. Three studies reported no change to ABI between groups (Barc 2006; Lindeman 2018; Pignon 2017). Li 2013 reported greater improvement in ABI in response to BMMNCs compared to placebo (P = 0.002). We downgraded the certainty of the evidence to very low due to risk of bias, imprecision, and inconsistency.

Only Lindeman 2018 reported pain‐free walking distance (PFWD), and results did not differ between the BMMNC and control groups. We downgraded the certainty of the evidence for this outcome to low due to imprecision and inconsistency.

All studies reported on side effects, and our analysis showed no clear difference in the numbers of side effects between BMMNC and control groups. We downgraded the certainty of the evidence to very low due to risk of bias, imprecision, and inconsistency.

The results of our review are limited due to the small number of randomised controlled trials (RCTs) meeting our inclusion criteria and differences in the control arms. The certainty of the evidence presented is very low to low, and we cannot draw any strong conclusions on the efficacy of BMMNC‐based therapy for CLI patients.

Overall completeness and applicability of evidence

The four included trials involved a very small number of participants, and there were substantial variations in the treatment strategies, control groups, and follow‐up duration, which limited our ability to combine the findings of these trials. Moreover, the use of different control groups in all studies may not be applicable to a much broader spectrum of CLI patients and their clinicians, and resulted in heterogeneity across the included studies. Given that only four studies with low numbers of participants were eventually included, significant concern exists on the completeness and applicability of the evidence presented in this review. Further studies with larger sample sizes are needed to permit a definitive conclusion. Future trials need to address important outcomes such as the role of treatment in the relief of pain, which can be beneficial in improving a patient's quality of life, as well as understanding the possible adverse effects of treatment. Furthermore, focusing on the comparison of different routes of administration, types of cells, and supportive bioengineered matrices could provide fundamental information to establish the effectiveness and safety assessment of the method. Moreover, specific studies about the implantation of stem cells in ischaemic tissues at an earlier stage of peripheral arterial disease are urgently needed to clarify how it can affect the course of this disabling disease.

Quality of the evidence

Overall, we judged the certainty of the evidence as very low (Table 1). Major reasons for downgrading the certainty of the evidence were concerns related to risk of bias, imprecision, and inconsistency. Methodological limitations included unclear randomisation methods and allocation concealment, as well as lack of blinding of participants and insufficient outcome data. Based on the available data, two studies did not adequately report random sequence generation or allocation concealment, putting them at risk of selection bias, and provided insufficient information regarding the blinding of participants (Barc 2006; Li 2013). We assessed two studies as at an overall low risk of bias (Lindeman 2018; Pignon 2017). We also downgraded the certainty of the evidence for imprecision of effect estimates due to the small numbers of studies with limited participants that contributed to all outcomes (the total number of participants from four trials was fewer than 200 participants), and for inconsistency of the results (due to wide confidence intervals across analyses and differences in the control groups). Statistical heterogeneity greatly affected our ability to answer our review question, and has impacted the validity of our conclusion. There is a need for high‐quality studies with larger sample sizes to assess the effects of the treatment regimen of cell‐based therapy for CLI in clinical practice. The data obtained from these studies are currently not of sufficient certainty to draw robust conclusions for the outcomes evaluated in this review regarding the use of BMMNC for the treatment of CLI.

Potential biases in the review process

An extensive literature search was performed by Cochrane Vascular. Two review authors independently determined study eligibility, and two review authors independently extracted data and performed quality assessment in order to reduce bias and subjectivity. There were no significant disagreements during the review process. We included only randomised clinical trials in our review. We are confident that all potential sources of data to be included in this review were carefully vetted.

Agreements and disagreements with other studies or reviews

To date, limited published RCTs have directly evaluated the effectiveness of intramuscular implantation of cell‐based products derived from bone marrow and compared it with conventional treatment options for the management of CLI. Moreover, these studies have yielded inconsistent results in terms of effectiveness. There are non‐RCT studies that have investigated the role of intramuscular implantation of various cell types (Franz 2009; Iso 2010). These studies have shown promising results: some studies have reported improved outcomes with intramuscular implantations, whilst few studies have not shown this approach to be beneficial for people with CLI (Dong 2013; Lara‐Hernandez 2010). A systematic review reported that although non‐RCTs suggest a benefit of cell treatment (lower the risk of amputation by 37%, increased amputation‐free survival by 18%, and improved wound healing by 59%, without affecting mortality), controlled trial studies with a low risk of bias do not support the promising results of these studies thus far (Rigato 2017). Rigato 2017 also reported that cell therapy with peripheral blood mononuclear cells (PBMNCs), but not other cell types, was associated with a significant improvement in amputation and amputation‐free survival, whereas only BMMNCs significantly improved wound healing. On the other hand, both BMMNCs and PBMNCs significantly improved ABI, transcutaneous pressure of oxygen (TcPO₂), and rest pain scores.

This update includes three new trials, bringing the total number of participants to 176. Although additional data are included, the findings presented in this update are consistent with the results of the two previous versions of this systematic review (Moazzami 2011; Moazzami 2014).

Previous meta‐analyses have shown promising results of stem cell‐based therapies versus conventional treatments in the management of CLI patients (Liu 2012; Teraa 2013; Wen 2011). In contrast to our review, these studies pooled and analysed outcome data of patients treated with cells from different sources and different types of cell administration techniques (BMMNCs, bone marrow mesenchymal stem cells, mobilised peripheral blood stem cells, Ixmyelocel‐T) and collectively reported them as bone marrow‐derived cell therapy. Another systematic review and meta‐analysis concluded that cell‐based therapy significantly reduced the amputation rate and improved ABI, as well as ulcer healing, when compared to non‐cell treatment groups (Liu 2015). Contrary to our review, Liu 2015 combined and analysed non‐randomised studies in their meta‐analysis. Consequently, in these meta‐analyses, the use of various types of cell products for implantation, as well as differences in routes of administration, may have had a profound impact on clinical outcomes and the generalisability of the conclusion.

A recent Cochrane Review involving seven RCTs with a total of 359 participants compared the efficacy and safety of autologous cells derived from different sources, prepared using different protocols, administered at different doses, and delivered via different routes for the treatment of 'no‐option' CLI patients (Abdul Wahid 2018). Similarly, pooled analyses did not show a clear difference in clinical outcomes between different stem cell sources and different treatment regimens of autologous cell implantation, and no strong conclusions were possible (Abdul Wahid 2018).

Authors' conclusions

Implications for practice.

We identified a small number of studies that met our inclusion criteria, and these differed in controls used and how they measured important outcomes. Limited data from the published trials provide very low‐ and low‐certainty evidence, and we are unable to draw conclusions that support the use of local intramuscular transplantation of bone marrow mononuclear cells (BMMNCs) for improving clinical outcomes in people with critical limb ischaemia.

Implications for research.

Further well‐conducted randomised double‐blind trials with high‐quality methodological assessments should be performed. Key outcomes of these new studies should be amputation‐free survival, angiographic analysis, assessment of pain reduction, and assessments of side effects and complications of the treatment. Sufficient numbers of participants should be included to provide statistically powerful information. Also, studies should be conducted in order to determine factors including the optimal dose of BMMNCs infused, the route of cell delivery, and the exact mechanism of action of the intervention. Moreover, the effects of implantation of other cell types and comparisons between them, as well as other routes of administration, should be addressed. Finally, longer durations of follow‐up and standardisation of outcome assessment methods are needed in future studies.

What's new

Date	Event	Description
19 July 2022	Amended	Spelling mistake in Plain Language Summary corrected.

History

Protocol first published: Issue 2, 2010
Review first published: Issue 12, 2011

Date	Event	Description
18 January 2022	New search has been performed	Search updated. Three new studies included, 38 additional studies excluded, and three new studies assessed as ongoing.
18 January 2022	New citation required but conclusions have not changed	Search updated. Three new studies included, 38 additional studies excluded, and three new studies assessed as ongoing. New authors joined the review team. Text updated and summary of findings table included. No change to conclusions
2 October 2014	New search has been performed	Search rerun. No new studies included. Thirty additional studies excluded.
2 October 2014	New citation required but conclusions have not changed	Search rerun. No new studies included. Thirty additional studies excluded. Minor edits made to the review text. New authors have joined the review team. No change to conclusions.

Appendices

Appendix 1. Databases searched and strategies used

Source	Search strategy	Hits retrieved
VASCULAR REGISTER IN CRSW	#1 Ischemia OR Limb Salvage OR Peripheral Vascular Diseases OR Arteriosclerosis OR Atherosclerosis OR Arterial Occlusive Diseases AND INREGISTER AND 01/01/2014_TO_19/03/2019:CRSINCENTRAL #2 mononuclear OR Transplantation, Autologous OR Bone Marrow Transplantation OR Leukocytes, Mononuclear AND INREGISTER AND 01/01/2014_TO_19/03/2019:CRSINCENTRAL #3 #1 AND #2	March 2019: 19 Nov 2021: 7
CENTRAL via CRSO	#1 MESH DESCRIPTOR Arteriosclerosis 948 #2 MESH DESCRIPTOR Limb Salvage EXPLODE ALL TREES 84 #3 MESH DESCRIPTOR Arteriosclerosis Obliterans 81 #4 MESH DESCRIPTOR Atherosclerosis 1095 #5 MESH DESCRIPTOR Arterial Occlusive Diseases 832 #6 MESH DESCRIPTOR Intermittent Claudication 839 #7 MESH DESCRIPTOR Ischemia 1609 #8 MESH DESCRIPTOR Peripheral Vascular Diseases 724 #9 (atherosclero* or arteriosclero* or PVD or PAOD or PAD):TI,AB,KY 12346 #10 ((arter* or vascular or vein* or veno* or peripher*) adj (occlus* or steno* or obstruct* or lesio* or block*)):TI,AB,KY 11683 #11 (peripheral adj3 dis*):TI,AB,KY 4966 #12 claudic*:TI,AB,KY 1905 #13 (isch* or CLI):TI,AB,KY 32862 #14 #1 OR #2 OR #3 OR #4 OR #5 OR #6 OR #7 OR #8 OR #9 OR #10 OR #11 OR #12 OR #13 54577 #15 MESH DESCRIPTOR Transplantation, Autologous EXPLODE ALL TREES 1487 #16 MESH DESCRIPTOR Bone Marrow Transplantation EXPLODE ALL TREES 1343 #17 MESH DESCRIPTOR Leukocytes, Mononuclear EXPLODE ALL TREES 6575 #18 mononuclear:TI,AB,KY 4087 #19 (bone adj marrow):TI,AB,KY 9072 #20 (autologous adj cell*):TI,AB,KY 3950 #21 BMC:TI,AB,KY 749 #22 #15 OR #16 OR #17 OR #18 OR #19 OR #20 OR #21 21528 #23 #14 AND #22 1086	March 2019: 678 Nov 2021: 428
Clinicaltrials.gov	Ischemia OR Limb Salvage OR Peripheral Vascular Diseases OR Arteriosclerosis OR Atherosclerosis OR Arterial Occlusive Diseases | mononuclear OR Transplantation, Autologous OR Bone Marrow Transplantation OR Leukocytes, Mononuclear |	March 2019: 28 Nov 2021: 7
ICTRP Search Portal	Ischemia OR Limb Salvage OR Peripheral Vascular Diseases OR Arteriosclerosis OR Atherosclerosis OR Arterial Occlusive Diseases | mononuclear OR Transplantation, Autologous OR Bone Marrow Transplantation OR Leukocytes, Mononuclear	March 2019: 6 Nov 2021: 4
MEDLINE (Ovid MEDLINE® Epub Ahead of Print, In‐Process & Other Non‐Indexed Citations, Ovid MEDLINE® Daily and Ovid MEDLINE®) 1946 to present	1 Arteriosclerosis/ 2 exp Limb Salvage/ 3 Arteriosclerosis Obliterans/ 4 Atherosclerosis/ 5 Arterial Occlusive Diseases/ 6 Intermittent Claudication/ 7 Ischemia/ 8 Peripheral Vascular Diseases/ 9 (atherosclero* or arteriosclero* or PVD or PAOD or PAD).ti,ab. 10 ((arter* or vascular or vein* or veno* or peripher*) adj (occlus* or steno* or obstruct* or lesio* or block*)).ti,ab. 11 (peripheral adj3 dis*).ti,ab. 12 claudic*.ti,ab. 13 (isch* or CLI).ti,ab. 14 or/1‐13 15 exp Transplantation, Autologous/ 16 exp Bone Marrow Transplantation/ 17 exp Leukocytes, Mononuclear/ 18 mononuclear.ti,ab. 19 (bone adj marrow).ti,ab. 20 (autologous adj cell*).ti,ab. 21 BMC.ti,ab. 22 or/15‐21 23 14 and 22 24 randomized controlled trial.pt. 25 controlled clinical trial.pt. 26 randomized.ab. 27 placebo.ab. 28 drug therapy.fs. 29 randomly.ab. 30 trial.ab. 31 groups.ab. 32 or/24‐31 33 exp animals/ not humans.sh. 34 32 not 33 35 23 and 34	March 2019: 340 Nov 2021: 563
Embase via OVID	1 arteriosclerosis/ 2 exp limb salvage/ 3 peripheral occlusive artery disease/ 4 atherosclerosis/ 5 intermittent claudication/ 6 ischemia/ 7 peripheral vascular disease/ 8 (atherosclero* or arteriosclero* or PVD or PAOD or PAD).ti,ab. 9 ((arter* or vascular or vein* or veno* or peripher*) adj (occlus* or steno* or obstruct* or lesio* or block*)).ti,ab. 10 (peripheral adj3 dis*).ti,ab. 11 claudic*.ti,ab. 12 (isch* or CLI).ti,ab. 13 or/1‐12 14 exp autotransplantation/ 15 exp bone marrow transplantation/ 16 exp mononuclear cell/ 17 mononuclear.ti,ab. 18 (bone adj marrow).ti,ab. 19 (autologous adj cell*).ti,ab. 20 BMC.ti,ab. 21 or/14‐20 22 13 and 21 23 randomized controlled trial/ 24 controlled clinical trial/ 25 random$.ti,ab. 26 randomization/ 27 intermethod comparison/ 28 placebo.ti,ab. 29 (compare or compared or comparison).ti. 30 ((evaluated or evaluate or evaluating or assessed or assess) and (compare or compared or comparing or comparison)).ab. 31 (open adj label).ti,ab. 32 ((double or single or doubly or singly) adj (blind or blinded or blindly)).ti,ab. 33 double blind procedure/ 34 parallel group$1.ti,ab. 35 (crossover or cross over).ti,ab. 36 ((assign$ or match or matched or allocation) adj5 (alternate or group$1 or intervention$1 or patient$1 or subject$1 or participant$1)).ti,ab. 37 (assigned or allocated).ti,ab. 38 (controlled adj7 (study or design or trial)).ti,ab. 39 (volunteer or volunteers).ti,ab. 40 trial.ti. 41 or/23‐40 42 22 and 41	March 2019: 1357 Nov 2021: 1842
CINAHL via EBSCO	S37 S21 AND S36 S36 S22 OR S23 OR S24 OR S25 OR S26 OR S27 OR S28 OR S29 OR S30 OR S31 OR S32 OR S33 OR S34 OR S35 S35 MH "Random Assignment" S34 MH "Triple‐Blind Studies" S33 MH "Double‐Blind Studies" S32 MH "Single‐Blind Studies" S31 MH "Crossover Design" S30 MH "Factorial Design" S29 MH "Placebos" S28 MH "Clinical Trials" S27 TX "multi‐centre study" OR "multi‐center study" OR "multicentre study" OR "multicenter study" OR "multi‐site study" S26 TX crossover OR "cross‐over" S25 AB placebo* S24 TX random* S23 TX trial* S22 TX "latin square" S21 S13 AND S20 S20 S14 OR S15 OR S16 OR S17 OR S18 OR S19 S19 TX autologous n cell* S18 TX bone n marrow S17 TX mononuclear S16 (MH "Leukocytes, Mononuclear+") S15 (MH "Bone Marrow Transplantation+") S14 (MH "Bone Marrow Transplantation, Autologous") S13 S1 OR S2 OR S3 OR S4 OR S5 OR S6 OR S7 OR S8 OR S9 OR S10 OR S11 OR S12 S12 TX isch* or CLI S11 TX claudic* S10 TX peripheral n3 dis* S9 TX (arter* or vascular or vein* or veno* or peripher*) n (occlus* or steno* or obstruct* or lesio* or block*) S8 TX atherosclero* or arteriosclero* or PVD or PAOD or PAD S7 (MH "Peripheral Vascular Diseases") S6 (MH "Ischemia") S5 (MH "Intermittent Claudication") S4 (MH "Arterial Occlusive Diseases") S3 (MH "Atherosclerosis") S2 (MH "Limb Salvage") S1 (MH "Arteriosclerosis")	March 2019: 37 Nov 2021: 40
AMED via OVID	1 Arteriosclerosis/ 2 exp Limb Salvage/ 3 Atherosclerosis/ 4 Intermittent Claudication/ 5 Ischemia/ 6 (atherosclero* or arteriosclero* or PVD or PAOD or PAD).ti,ab. 7 ((arter* or vascular or vein* or veno* or peripher*) adj (occlus* or steno* or obstruct* or lesio* or block*)).ti,ab. 8 (peripheral adj3 dis*).ti,ab. 9 claudic*.ti,ab. 10 (isch* or CLI).ti,ab. 11 or/1‐10 12 mononuclear.ti,ab. 13 (bone adj marrow).ti,ab. 14 (autologous adj cell*).ti,ab. 15 BMC.ti,ab. 16 or/12‐15 17 11 and 16 18 exp CLINICAL TRIALS/ 19 RANDOM ALLOCATION/ 20 DOUBLE BLIND METHOD/ 21 Clinical trial.pt. 22 (clinic* adj trial*).tw. 23 ((singl* or doubl* or trebl* or tripl*) adj (blind* or mask*)).tw. 24 PLACEBOS/ 25 placebo*.tw. 26 random*.tw. 27 PROSPECTIVE STUDIES/ 28 or/18‐27 29 17 and 28 30 ("2017" or "2018" or "2019").yr. 31 29 and 30	March 2019: 0 Nov 2021: 0

Data and analyses

Comparison 1. BMMNC versus control.

Outcome or subgroup title	No. of studies	No. of participants	Statistical method	Effect size
1.1 All‐cause mortality	3	123	Risk Ratio (M‐H, Random, 95% CI)	1.00 [0.15, 6.63]
1.2 Amputation	4	176	Risk Ratio (M‐H, Random, 95% CI)	0.52 [0.27, 0.99]
1.3 Amputation ‐ sensitivity analysis	2	89	Risk Ratio (M‐H, Random, 95% CI)	0.52 [0.19, 1.39]
1.4 Side effects and complications	4	176	Risk Ratio (M‐H, Random, 95% CI)	2.13 [0.50, 8.97]
1.5 Side effects and complications ‐ sensitivity analysis	2	89	Risk Ratio (M‐H, Random, 95% CI)	2.69 [0.11, 63.18]

Characteristics of studies

Characteristics of included studies [ordered by study ID]

Barc 2006.

Study characteristics
Methods	Study design: stated as randomised Method of randomisation: not stated Blinding: not stated Exclusions postrandomisation: not stated Losses to follow‐up: not stated
Participants	Country: Poland Participants: 29 randomised Mean age: not stated Sex: not stated Inclusion criteria: patients with CLI with risk of amputation presence of rest pain and/or necrosis lasting > 12 weeks no progress after 8 weeks of conventional therapy ABI < 0.5 in 2 independent examinations performed during the 7‐day interval pre‐inclusion peripheral type of atherosclerosis and no possibility for operative therapy, confirmed in angiogram Exclusion criteria: age < 18 years need for urgent amputation reasons for ischaemia other than atherosclerosis cancer absence of conscious consent lack of understanding of the idea of the therapy by patient poor general condition (life expectancy < 6 months)
Interventions	Treatment group: type of cells: BMMNC cell isolation: not mentioned dose of cells: not reported route and frequency of delivery: multiple IM injections Control group: not stated in the paper, but described by the authors as standard conservative therapy
Outcomes	Primary outcomes: ABI photographic documentation of ischaemic ulcerations and necrosis subjective parameters (feeling of pain in VAS, QoL in WHO scale) Secondary outcomes: not stated Outcome assessment points: baseline and months 1, 3, and 6
Funding	Not reported
Declarations of interest	Not reported
Notes	It is unclear who injects monocytes.
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Unclear risk	The study is described as randomised, but details are unclear.
Allocation concealment (selection bias)	Unclear risk	Not mentioned
Blinding of participants and personnel (performance bias) All outcomes	Unclear risk	Not mentioned
Blinding of outcome assessment (detection bias) All outcomes	Unclear risk	Not mentioned
Incomplete outcome data (attrition bias) All outcomes	Unclear risk	All participants are accounted for.
Selective reporting (reporting bias)	High risk	The ABI and pain data were not reported in sufficient detail to include in the analysis.
Other bias	Low risk	We did not identify any other potential sources of bias.

Li 2013.

Study characteristics
Methods	Study design: stated as randomised Method of randomisation: not stated Blinding: single‐blind Exclusions postrandomisation: stated that 16 patients could not be evaluated for haemodynamic assessment because of extensive ulceration that made ankle pressure assessment not feasible Losses to follow‐up: not stated
Participants	Country: China Participants: 58 randomised Mean age: 61 years in treatment group, 63 years in control group Sex: male and female Inclusion criteria: evidence of CLI including rest pain and/or non‐healing ischaemic ulcers for a minimum of 4 weeks without improvement in response to conventional therapies Exclusion criteria: history of malignancy evidence of possible malignancies after evaluation with carcinoembryonic antigen levels, chest radiographs, CT scans, and mammography in women or prostate examination in men
Interventions	Treatment group: type of cells: BMMNC cell isolation: cells were extracted from the posterior superior iliac spine. BMMNCs were isolated from the bone marrow by density gradient centrifugation with lymphocyte separating fluid. Cell counting was performed. Isolated BMMNCs were diluted into 50 to 120 mL suspensions to be transplanted. dose of cells: 1 × 107 piece [sic]/mL BMMNC transplant route and frequency of delivery: multiple IM injections Control group: 0.9% sodium chloride (saline)
Outcomes	Primary outcomes: safety assessments included adverse events, physical examination, cancer screening, ECG, blood chemistry, haematology, and urinalysis for fever, allergies, myocardial, stoke, malignancy, and death Secondary outcomes: haemodynamic improvement (an absolute increase of > 15% in the ABI) skin ulcers improvement using photographic documentation of ischaemic ulcerations pain score improvement using VAS limb survival Outcome assessment points: baseline and months 1, 3, and 6
Funding	Not reported
Declarations of interest	Not reported
Notes	It is unclear who injects monocytes.
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Unclear risk	The study is described as randomised, but details are unclear.
Allocation concealment (selection bias)	Unclear risk	There was no information regarding allocation.
Blinding of participants and personnel (performance bias) All outcomes	Unclear risk	Quote: "This was a single blinded study aimed at analyzing the effect of..." Comment: not stated who was blinded
Blinding of outcome assessment (detection bias) All outcomes	Unclear risk	Quote: "This was a single blinded study aimed at analyzing the effect of..." Comment: not stated who was blinded
Incomplete outcome data (attrition bias) All outcomes	High risk	Quote: "Sixteen patients could not be evaluated for hemodynamic assessment because of extensive ulceration that made ankle pressure assessment not feasible" and "Fifteen patients were not evaluated for pain scores owing to minor surgical intervention just before the transplant or they could not understand the VAS scale"
Selective reporting (reporting bias)	Low risk	We did not identify any reporting bias.
Other bias	Low risk	We did not identify any other potential sources of bias.

Lindeman 2018.

Study characteristics
Methods	Study design: stated as randomised Method of randomisation: computer‐generated randomisations and allocation Blinding: fully blinded Exclusions postrandomisation: not stated Losses to follow‐up: 1 participant in the placebo group
Participants	Country: the Netherlands Participants: 54 randomised Mean age: 58.5 years in treatment group, 57.8 years in control group Sex: male and female Inclusion criteria: persistent disabling claudication (Fontaine’s stages IIb/III or Rutherford’s categories 3/4) or CLI (Fontaine’s stages IV or Rutherford’s categories 5/6) despite > 6 months optimal medical therapy (including exercise therapy, and revascularisation attempts in accordance with prevailing guidelines) Exclusion criteria: diabetes immune suppressive therapy age < 18 years compromised life expectancy (anticipated survival < 1 year) severe tissue loss or non‐manageable pain with an anticipated need for amputation within the first month of therapy
Interventions	Treatment group: type of cells: BMMNC cell isolation: BMMNCs were collected from multiple locations from the posterior iliac crests. The obtained bone marrow suspension was then filtered and concentrated to a final volume of 40 mL on the COBE Spectra Apheresis System (Gambro, Stockholm, Sweden) in the LUMC GMP‐facility without any further treatment. dose of cells: 1.7 × 109 total route and frequency of delivery: IM injections in 40 locations (1 mL per injection site) Control group: diluted autologous peripheral blood, which was visually indistinguishable from the BMMNC
Outcomes	Primary outcomes: PFWD or limb salvage full wound recovery Secondary outcomes: ABI SF‐36 Pain (BPI‐SF) Time points for assessment: months 1, 6, and 12
Funding	Not reported
Declarations of interest	Not reported
Notes	It is unclear who injects monocytes.
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Quote: "Computer randomization and allocation were done after bone marrow collection, by the stem laboratory in permuted blocks of size 4 in 2 strata (disabling claudication or CLI) and secured until all data entry was completed and the database was locked"
Allocation concealment (selection bias)	Low risk	Allocation was adequately concealed.
Blinding of participants and personnel (performance bias) All outcomes	Low risk	Quote: "All patients, clinicians, and trial investigators remained blinded for the treatment allocation until reaching the secondary 12 month end point."
Blinding of outcome assessment (detection bias) All outcomes	Low risk	Quote: "All patients, clinicians, and trial investigators remained blinded for the treatment allocation until reaching the secondary 12 month end point."
Incomplete outcome data (attrition bias) All outcomes	Low risk	All randomised participants were analysed, except for 1 participant in the placebo group. Quote: "One patient in the placebo group was unable to comply with the follow‐up (language barrier), hence 25 patients could be evaluated."
Selective reporting (reporting bias)	Low risk	The clinical measures prespecified in the protocols were adequately reported in the results.
Other bias	Low risk	None suspected.

Pignon 2017.

Study characteristics
Methods	Study design: stated as randomised Method of randomisation: website‐generated randomisation Blinding: double‐blind Exclusions postrandomisation: not stated Intention‐to‐treat analysis: used Losses to follow‐up: 1 participant in placebo group and 1 participant in treatment group
Participants	Country: France Participants: 38 randomised Mean age: 72 years in treatment group, 65 years in control group Sex: male and female Inclusion criteria: age ≥ 18 years presented with atherosclerosis‐related CLI with no sign of improvement following previous appropriate medical treatment Exclusion criteria: Buerger’s disease active infection uncontrolled diabetes mellitus prior history of neoplasm or malignancy contraindication for general anaesthesia prothrombin time < 50% myocardial or brain infarction within 3 months any medical condition contraindicating the initiation of anticoagulation unexplained haematological abnormality HIV hepatitis B or C virus infection any concomitant disease associated with a life expectancy of < 1 year
Interventions	Treatment group: type of cells: BMMNC cell isolation: 500 mL of bone marrow collected from the posterior iliac crests. 8 centres used a blood‐cell separator (COBE Spectra, version 4, Bone Marrow Processing Program, Gambro BCT, Lakewood, CO, USA); 2 centres used a blood‐cell separator requiring a Ficoll density‐gradient for isolation of the BMMNC. dose of cells: 1.3 × 109 total (not clear in report) route and frequency of delivery: IM injections (30 intramuscular injections of 1 mL) Control group: 30 mL saline with 4 mL autologous peripheral blood
Outcomes	Primary outcomes: major amputation rate mortality Secondary outcomes: ABI TcPO2 ulcers pain Time points for assessment: baseline, days 1, 3, 15, 28, and then months 6 and 12
Funding	The reported work was supported by the French Ministry of Health (Programme Hospitalier de Recherche Clinique 2007) and by the French Blood Agency.
Declarations of interest	Not reported
Notes	It is unclear who injects monocytes.
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Quote: "Randomization was performed by the local investigator in charge of the patient, through a dedicated website, with stratification by center."
Allocation concealment (selection bias)	Low risk	Allocation was adequately concealed.
Blinding of participants and personnel (performance bias) All outcomes	Low risk	Quote: "The steering committee, the investigators, the observers, and the patients were unaware of the treatment allocation."
Blinding of outcome assessment (detection bias) All outcomes	Low risk	Quote: "The steering committee, the investigators, the observers, and the patients were unaware of the treatment allocation."
Incomplete outcome data (attrition bias) All outcomes	Low risk	2 participants (1 in placebo group and 1 in treatment group) dropped out of study due to adverse events. Quote: "Therefore, the modified ITT analysis excluded these 2 patients and the analysis was conducted on 19 patients in the placebo group and 17 in the BMMNC group."
Selective reporting (reporting bias)	High risk	The data for ABI and pain were not reported in sufficient detail to include in the analysis.
Other bias	Low risk	None suspected.

ABI: ankle‐brachial index
BMMNC: bone marrow mononuclear cell
BPI‐SF: Brief Pain Inventory‐Short Form
CLI: critical limb ischaemia
CT: computed tomography
ECG: echocardiographs
IM: intramuscular
PFWD: pain‐free walking distance
QoL: quality of life
SF‐36: 36‐Item Short‐Form Health Survey
TcPO₂: transcutaneous pressure of oxygen
VAS: visual analogue scale
VEGF: vascular endothelial growth factor
WHO: World Health Organization

Characteristics of excluded studies [ordered by study ID]

Study	Reason for exclusion
Benoit 2011	This study used bone marrow from the iliac crest, but after aspiration, BMMNC was processed into 40 mL of concentrate using the SmartPReP2 Bone Marrow Aspirate Concentrate (BMAC) system.
BONMOT 2008	The study is an ongoing RCT investigating the effects of autologous bone marrow concentrate, not BMMNCs.
Burt 2010	This study assessed the safety and feasibility of autologous CD133+ cells and included no control group.
Capiod 2009	The study compared the effect of transplantation of BMMNCs or G‐CSF‐mobilised PBMNCs, and included no control group.
Chen 2009	The study included people with diabetic foot not CLI, and participants received autologous bone marrow MSC, not mononuclear cells.
Debin 2008	The study investigated the effects of bone marrow MSC, not BMMNCs.
Dong 2013	The study investigated the effects of purified CD34+ cells, not BMMNCs.
Dong 2018	The study compared the effect of transplantation of PBMNCs or purified CD34+ cells, and included no control group.
Du 2017	The study assessed the clinical efficacy of implanted umbilical cord MSCs combined with bone marrow stem cells compared with implanted bone marrow stem cells for treatment of lower limb ischaemia.
Flugelman 2017	The study assessed venous endothelial cells combined with venous smooth muscle cells.
Frogel 2017	This is a pilot open‐label study of treatment progenitor cells derived from peripheral blood for people with CLI.
Gupta 2013	The study investigated the effects of allogeneic bone marrow‐derived MSC, not BMMNC.
Gurunathan 2009	The study is an ongoing RCT investigating the effects of autologous bone marrow concentrate, not BMMNCs.
Higashi 2010	The study investigated the combined effect of BMMNC implantation and sarpogrelate, a selective 5‐HT(2A) antagonist.
Holzinger 1994	The study investigated people with chronic skin ulcers caused by chronic arterial occlusive disease or venous post‐thrombotic syndrome, not CLI.
Horie 2018	This RCT evaluated the efficacy and safety of G‐CSF‐mobilised PBMNC transplantation in people with PAD.
Huang 2005a	The study investigated PBMNC, not BMMNC.
Iafrati 2011	The study investigated BMA concentrate, not BMMNC.
Iafrati 2016	The study investigated BMA concentrate, not BMMNC.
JUVENTAS 2008	The study is an ongoing RCT investigating the effects of IA injection of BMMNCs.
Kirana 2007	The study investigated people with diabetic foot ulcers, not CLI.
Kirana 2012	The study investigated people with diabetic foot ulcers, not CLI.
Klepanec 2012	The study compared IA and IM MSC therapy with no control group.
Korymasov 2009	The study is a randomised triple‐arm study investigating the safety and effectiveness of highly purified CD133+ autologous stem cells in CLI.
Lasala 2011	The study investigated the transplantation of an autologous bone marrow‐derived combination stem cell product.
Lu 2011	The study compared bone marrow MSC with BMMNCs with no control group.
Madaric 2011	The study compared IA and IM application of autologous BMC transplantation.
Majumdar 2015	This phase 2 study assessed allogeneic MSC in CLI.
Mohamed 2020	The study included bone marrow mesenchymal stromal cell therapy.
Molavi 2016	This RCT investigated the safety and efficacy of repeated BMMNC injections in comparison with a single BMMNC injection in CLI patients.
Murphy 2017	This double‐blinded, placebo‐controlled trial assessed the safety and efficacy of autologous concentrated BMA versus placebo.
NCT00282646	This is an ongoing RCT investigating the effects of IA injection of autologous BMMNCs versus placebo.
NCT00306085	The study investigated patients with peripheral atherosclerosis, not CLI.
NCT00498069	This is an ongoing RCT investigating the effects of BMCs, not BMMNCs.
NCT00539266	This is an ongoing RCT investigating the effects of BMCs, not BMMNCs.
NCT00595257	This is an ongoing RCT investigating the effects of autologous bone marrow concentrate, not BMMNCs.
NCT00616980	This is an ongoing RCT investigating the effects of peripheral blood‐derived stem cells, not BMMNCs.
NCT00913900	The study is an ongoing RCT investigating the effects of adult stem cells.
NCT00922389	This is an ongoing RCT investigating the effects of CD34 positive cells, not BMMNCs.
NCT00955669	This is an ongoing RCT comparing autologous MSC and mononuclear cells, not BMMNCs.
NCT01049919	This is an ongoing RCT investigating the effects of autologous concentrated BMA, not BMMNCs.
NCT01245335	This is an ongoing RCT investigating the effects of BMA concentrate, not BMMNCs.
NCT01584986	This RCT assessed the safety and efficacy of autologous ACPs compared with SMT.
NCT02336646	This RCT compared allogeneic MSCs versus placebo in people with CLI.
NCT03174522	This clinical trial compared Rexmyelocel‐T versus placebo in people with CLI.
NCT03214887	This clinical trial investigated the safety and efficacy of hyaluronan combined with BMMNCs for PAD.
NCT03304821	This double‐blind, placebo‐controlled RCT examined whether 3 weeks of 3‐times‐a week injection of GM‐CSF would improve measures of ischaemia in people with IC compared with placebo.
NCT03339973	This clinical trial is investigating the efficacy and safety of 1 dose of allo‐APZ2‐PAOD administered IM into the affected lower leg of people with PAD.
Niven 2017	This pilot open‐label study examined treatment with progenitor cells for PAD.
Ohtake 2017	This prospective phase 1/2 interventional clinical trial examined autologous G‐CSF‐mobilised CD34+ cell transplantation in haemodialysis patients with CLI.
Pawan 2012	The study investigated adult bone marrow‐derived allogeneic MSC.
Perin 2011	The study investigated autologous therapy with bone marrow‐derived aldehyde dehydrogenase bright cells. These cells are derived from CD15 and glycophorin A‐expressing cells from autologous bone marrow via immunomagnetic beads and cell sorters.
Perin 2017	The study investigated autologous therapy with bone marrow‐derived aldehyde dehydrogenase bright cells. These cells are derived from CD15 and glycophorin A‐expressing cells from autologous bone marrow via immunomagnetic beads and cell sorters.
Poole 2013	This trial investigated the effects of GM‐CSF, not cell therapy, in people with IC.
Prochazka 2010	This RCT investigated major limb amputation in participants given autologous BMC compared to those given standard care for CLI and foot ulcer.
PROVASA 2011	This is a multicentre, phase II, double‐blind RCT comparing IA administration of BMMNC or placebo followed by active treatment with BMMNC (open‐label) after 3 months.
RESTORE‐CLI 2012	This is an ongoing RCT investigating the effects of vascular repair cells, not BMMNCs.
Sharma 2021	In this study, BMMNC was injected IA, not IM.
Skóra 2015	This study investigated autologous BMMNC plus VEGF, not BMMNC alone.
Subramaniyam 2009	The study investigated patients with IC, not CLI.
Szabo 2013	The study investigated the effects of autologous stem cell therapy, not BMMNCs.
Teraa 2015	This double‐blind, placebo‐controlled RCT compared BMMNCs versus placebo in people with limb ischaemia, which was administered IA.
Tournois 2015	This phase 1 and 2 clinical trial examined BMC therapy products and cell therapy products obtained by cytapheresis (peripheral blood‐cell therapy products).
Walter 2011	The study compared the effect of IA route of transplantation.
Wang 2014	This trial assessed the efficacy and safety of the combination of peripheral blood mononuclear cells and Panax notoginseng saponins for treatment of CLI.
Wang 2017	This multicentre, double‐blind, placebo‐controlled RCT assessed the efficacy of IM injections of concentrated BMA for promoting amputation‐free survival in people with poor‐option CLI.
Wang 2018	This study compared IM injections of allogeneic MSCs or autogenous concentrated BMA.
Wijnand 2018	This study used allogeneic MSC for CLI.
Zafarghandi 2010	The study compared the effect of transplantation of BMMNCs with G‐CSF‐mobilised PBMNCs and included no control group.
Zhang 2010	The study only included people with diabetes.
Zhao 2008	The study investigated the effect of combined PBMNCs and BMMNCs injection versus placebo.
Zhou 2017a	This open, parallel‐control RCT compared BMMNC with SMT.
Zhou 2017b	This prospective, single‐centre, open‐label RCT compared peripheral blood CD34+ cells transfected with VEGF‐165 versus SMT.

ACPs: angiogenic cell precursors
BMA: bone marrow aspirate
BMC: bone marrow cell
BMMNC: bone marrow mononuclear cell
CLI: critical limb ischaemia
G‐CSF: granulocyte colony‐stimulating factor
GM‐CSF: granulocyte‐macrophage colony‐stimulating factor
IA: intra‐arterial
IC: intermittent claudication
IM: intramuscular
MSC: mesenchymal stem cells
PAD: peripheral arterial disease
PBMNC: peripheral blood‐derived mononuclear cell
RCT: randomised controlled trial
SMT: standard medical treatment
VEGF: vascular endothelial growth factor

Characteristics of ongoing studies [ordered by study ID]

NCT00753025.

Study name	Autologous bone marrow for lower extremity ischaemia treating
Methods	Allocation: randomised Endpoint classification: safety/efficacy study Intervention model: parallel assignment Masking: quadruple (participant, care provider, investigator, outcomes assessor) Primary purpose: treatment
Participants	Inclusion criteria: obliterating lower extremity atherosclerosis IIB stage (on Fontaine classification) PFWD of 10 to 50 m pulse absence on dorsalis pedis, tibialis posterior, poplitea absence of ischaemia in rest and necrotic changes mainly distal form of disease (lesion of a superficial femoral artery, a popliteal artery, anticnemion (anterior edge of the tibia) arteries) according to an angiography that testifies to impossibility of reconstructive operation performance after lumbar sympathectomy and tibial bone osteoperforations executed previously heavy smokers Exclusion criteria: insulin‐dependent diabetes myocardial infarction or stroke within the past year idiopathic hypertension III stage anaemia and other diseases of blood decompensation of chronic diseases that are contraindications to any surgical operation HIV infection A virus hepatitis oncological disease prior history of chemotherapy
Interventions	Biological: injection of isolated CD133+ cells
Outcomes	Primary outcome measure: increase in PFWD
Starting date	September 2008
Contact information	Andrey Toropovskiy, Clinical Center of Cellular Technologies, Russia
Notes	Primary completion date: September 2008

NCT01446055.

Study name	Safety and efficacy study of autologous BMMNC processed by two methods for treating patients with CLI
Methods	Allocation: randomised Endpoint classification: safety/efficacy study Intervention model: parallel assignment Masking: single‐blind (participant) Primary purpose: treatment
Participants	Inclusion criteria: Fontaine stage 2 to 4 or resting ABI < 0.7 age between 20 and 80 years signed informed consent, voluntary participants diagnosis of lower extremity arterial occlusive disease, or diabetic lower limb ischaemia, or Buerger’s disease Exclusion criteria: poorly controlled diabetes (glycated haemoglobin > 7.0%) and proliferative retinopathy (III to IV stage) malignancy history in the past 5 years or serum level of tumour markers elevated more than doubled severe heart, liver, kidney, respiratory failure or poor general condition and cannot tolerate BMMNC implantation serious infections (such as cellulitis, osteomyelitis, etc.) or gangrene such that a major amputation cannot be avoided aortic or iliac or common femoral artery occlusion pregnant female, or reproductive age female, who wants to give birth throughout the course of the study life expectancy less than 1 year
Interventions	Experimental: autologous BMMNC is enriched with ResQ process (an automatic cell separator). Then the cell product is implanted into the ischaemic limbs of a participant. Active comparator: a conventional method based on Ficoll cell separation is used to process bone marrow
Outcomes	Primary outcomes: cell treatment‐related adverse event: temperature, pulse, respiration, blood pressure, routine analysis of blood and urine, liver function (ALT, AST), renal function (blood urea nitrogen, creatinine), function of coagulation (APTT, prothrombin time, fibrinogen, thrombin time), ECG, local inflammatory response, cell treatment‐related death, cell treatment‐related unexpected amputation Secondary outcomes: ulcer size rest pain score cold sensation score claudication distance (m) resting ABI resting TcO (mmHg) collateral vessel score amputation rate skin microcirculation measurement resting TBI
Starting date	October 2011
Contact information	Contact: Yongquan Gu, MD, Xuanwu Hospital, Beijing; gu‐yq@263.net
Notes	The recruitment status of this study is unknown because the information has not been verified recently on ClinicalTrials.gov.

NCT02454231.

Study name	Monocentric trial: stem cell emergency life threatening limbs arteriopathy (SCELTA)
Methods	Allocation: randomised Endpoint classification: safety/efficacy study Intervention model: parallel assignment Masking: open‐label Primary purpose: treatment
Participants	Inclusion criteria: men and women older than 40 years of age with a diagnosis of CLI due to atherosclerosis of the lower extremities, as defined by the presence of persistent rest pain requiring systemic and continued analgesic treatment in the past 15 days and/or the presence of trophic lesions imputable to the occluding arteriopathy, ABI < 0.40 (with systolic ankle pressure < 50 to 70 mmHg), TBI < 0.40 (with big toe systolic pressure < 30 to 50 mmHg), and TcO < 30 mmHg eligible for treatment and enrolled only after demonstration that intravascular or surgical revascularisation was not possible, as revealed by echography and angio‐CAT, or when the patient refused to undergo surgical treatments and after written informed consent was obtained Exclusion criteria: age < 40 not atherosclerotic CLI myocardial infarction occurrence within 6 months cardiac failure of III‐IV class NYHA ejection fraction < 40% arterial hypertension (> 160/100 mmHg) uncontrolled despite usage of 2 antihypertensive drugs presence of current or chronic severe infectious disease osteomyelitis diabetes with glycated haemoglobin > 7.5 proliferative diabetic retinopathy haemorrhagic disorders non‐atherosclerotic arteriopathy chronic airway insufficiency (pO2 < 65 mmHg, pCO2 > 0.50 mmHg) renal failure (creatinine > 2 mg/dL) contraindications or intolerance to contrast media for radiological imaging
Interventions	Experimental: peripheral blood EPC injection Active comparator: BMMNC injection
Outcomes	Primary outcomes: safety as measured by evaluation of any adverse event temporarily correlated with treatment changes in ischaemic leg perfusion from baseline Secondary outcomes: improvement in mean values of TcO improvement in mean values of ABI improvement in vessel anatomical status improvement in leg perfusion improvement in vessel anatomical status quality of life improvement improvement in rest pain improvement in trophic limb lesions reduction in number of major amputations improvement in microvascular anatomy
Starting date	September 2009
Contact information	Contacts: Enrico Maggi, professor, University of Florence, Italy; enrico.maggi@unifi.it Francesco Annunziato, professor; francesco.annunziato@unifi.it
Notes	Primary completion date: May 2015

ABI: ankle‐brachial index
Angio‐CAT: computerised tomography (CT) coronary angiogram
APTT: activated partial thromboplastin time
ALT: alanine aminotransferase
AST: aspartate aminotransferase
BMI: body mass index
BMMNC: bone marrow mononuclear cells
CLI: critical limb ischaemia
DVT: deep vein thrombosis
ECG: electrocardiography
EPC: endothelial progenitor cells
G‐CSF: granulocyte colony‐stimulating factor
IC: intermittent claudication
IM: intramuscular
NYHA: New York Heart Association
PAD: peripheral arterial disease
pCO₂: partial pressure of carbon dioxide
PB‐MNC: peripheral blood mononuclear cells
PFWD: pain‐free walking distance
pO₂: partial pressure of oxygen
TASC: TransAtlantic Intersociety Consensus
TBI: toe brachial index
TcO: transcutaneous oxygen pressure

Differences between protocol and review

2022 version

For this update, we amended the title from 'Local intramuscular transplantation of autologous mononuclear cells for critical lower limb ischaemia' to 'Local intramuscular transplantation of autologous bone marrow mononuclear cells for critical lower limb ischaemia' to be consistent with our review objective. We reassessed previously included studies and previously excluded studies. We excluded a previously included study, Huang 2005a, as the intervention was not bone marrow mononuclear cells. We classified studies previously assessed as excluded because they were not randomised as not relevant, as recommended (Higgins 2021). We rephrased the outcomes 'progression of disease in terms of the incidence of amputation' and 'progression of disease in terms of incidence of surgical reconstruction or a non‐surgical (radiological) intervention' to 'incidence of amputation' and 'angiographic analysis', in order to improve readability and clarify outcomes relevant to the patient population. In keeping with current Cochrane recommendations, we also included a summary of findings table and assessed outcomes using the GRADE criteria.

2014 version

We assessed risk of bias using the Cochrane risk of bias tool, as described in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2011). In order to reflect clinical importance, we considered 'incidence of amputation' and 'increase in ankle‐brachial index (ABI)' as primary and secondary outcomes, respectively, in the final review.

Contributions of authors

BM: selected trials for inclusion, assessed methodological quality, extracted data, analysed data, and wrote the review
ZM: selected trials for inclusion, assessed methodological quality, extracted data, analysed data, and wrote the review
ZZ: selected trials for inclusion, assessed methodological quality, extracted data, analysed data, and wrote the review
EF: selected trials for inclusion, assessed methodological quality, extracted data, analysed data, and wrote the review
AR: selected trials for inclusion, assessed methodological quality, extracted data, analysed data, and wrote the review
ED: selected trials for inclusion, assessed methodological quality, extracted data, analysed data, and wrote the review
KM: conceived, designed, co‐ordinated, and wrote the protocol; selected trials for inclusion, assessed methodological quality, extracted data, analysed data, and wrote the review

Sources of support

Internal sources

• No sources of support provided

External sources

• Chief Scientist Office, Scottish Government Health Directorates, The Scottish Government, UK

  The Cochrane Vascular editorial base is supported by the Chief Scientist Office.

Declarations of interest

BM: none known
ZM: none known
ZZ: none known
EF: none known
AR: none known
ED: none known
KM: none known

Edited (no change to conclusions)