This clinical study was undertaken at Central Hospital of Wuhan, Hubei Province, China. All participants were briefly informed about this study. The informed consent was obtained during the study enrollment. This study was approved by the Ethics Committee of the Central Hospital of Wuhan, Wuhan, China.
Study design and participants
In this single-center clinical study, we investigated the clinical effect of BM-MNCs transplantation in 60 patients with refractory DPN recruited from March 2014 to December 2017. The detailed inclusion and exclusion criteria were listed below. All enrolled patients received the autologous BM-MNCs therapy and followed-up after transplantation for 2 days, 1, 4, 12, 24 and 48 weeks.
This clinical study also included 30 healthy control individuals (Control group) and 30 T2DM patients without complications (DM group). Age, sex, and BMI in all three groups were comparable (all p>0.05).
Inclusion criteria of DPN group
1) Age at 40 to 70 years; 2) T2DM patients defined by 2013 American Diabetes Association (ADA) standards ; 3) DPN was defined as the presence of an abnormality of nerve conduction and a symptom or a sign of neuropathy; 4) refractory DPN, defined as no significant relief of the neuropathic symptoms or signs when combine conventional drug therapies for at least one year, which include the antioxidant (a-lipoic acid), aldose reductase inhibitors and transketolase activators (thiamines and allithiamines).
Exclusion criteria of DPN group
1) Severe hepatic and renal dysfunctions; 2) hypercoagulable states or with hematological diseases; 3) foot ulcers and limb deformity; 4) pregnancy; 5) evidence of malignancy during the last 5 years; 6) life expectancy less than 6 months.
Preparation of BM-MNCs
The procedures used for the BM-MNCs treatment have been described in details previously . Briefly, patients were subcutaneously injected 5ug·kg-1·d-1 recombinant human granulocyte colony-stimulating factor (G-CSF, Qilu Pharmaceutical, China) for 3 consecutive days to mobilize the stem cells in bone marrow. After G-CSF mobilization, approximately 200-300mL bone marrow was harvested from the posterior superior iliac crest under anesthetic conditions in a sterile surgical environment.
The preparation of BM-MNCs was processed in the laminar flow laboratory. Mononuclear stem cells were isolated by Ficoll-Hypaque density–gradient centrifugation. Then, the mononuclear cell layer was harvested and washed 3 times with normal saline and resuspended in 50 mL normal saline. The total concentration of CD34+ cells in the cell suspension comprising mononuclear cells was calculated using flow cytometry.
The prepared BM-MNCs suspensions were injected intramuscularly to both thighs and legs of DPN patients (50 sites, 2 cm*2 cm in intervals, 1-1.5 cm in depth, 1 mL BM-MNCs per site) under continuous monitoring of vital parameters in a sterile surgical environment. Patients were followed-up for at least 24 hours in the intensive care unit after the injection.
Clinical assessment before and after transplantation
Clinical and laboratory data were collected before BM-MNCs administration and at every follow-up visit in DPN group. All patients received similar ordinary treatment throughout the course of this clinical study, including intensive control of blood glucose, blood pressure, and blood lipids. Smoking cessation was encouraged during the study. Attention was paid during the follow-up visits specifically to any potential adverse effects due to the transplantation.
Measurement of nerve conduction studies (NCS, Viking Quest®, Nicolet Biomedical Inc, WI, USA) was performed by the same experienced technician who was blinded to the patients’ clinical information according to validated standards. Routine NCS measurements were performed pre-transplantation and at 12, and 48 weeks post-transplantation. The observation items of NCS included sensory nerve conduction velocity (sNCV), sensory nerve action potential (SNAP) in superficial peroneal and sural nerves, motor nerve conduction velocity (mNCV), and compound muscle action potential (CMAP) in peroneal and posterior tibial nerves.
Neurological evaluations were performed with Toronto Clinical Scoring System (TCSS) before the therapy and at every follow up visit post transplantation.
Blood samples of all the subjects in the study were collected to test the levels of inflammatory markers and growth factors at baseline and at each post-transplantation follow-up visit in DPN group. Venous blood samples were collected from the subjects after a 12-h overnight fast. Serum were separated and stored at -70 ℃.Commercially available enzyme immunoassay (ELISA) kits were used to measure the inflammatory cytokines (IL-6, TNF-α, IL-10, sICAM-1) and growth factors (VEGF and NGF) according to the manufacturer’s instruction. All kits were provided by Quantikine, R&D Systems.
Outcome assessment of BM-MNCs transplantation
The primary endpoint of the study was the improvement of NCV. Responder to BM-MNCs therapy was defined as the sensory or motor nerve conduction velocity increased at least by 30% during the follow-up periods. Patients without obvious change of NCV were considered non-responders.
Measurement data were expressed as mean ± standard deviation (SD) for continuous variables, interquartile ranges for nonnormal data, and in percentages for discrete variables. Group differences for continuous variable were tested by independent t test or the Mann-Whitney U test. Dichotomous variables were analyzed with Fisher exact test. Multiple group comparisons were performed by analysis of variance (ANOVA). The comparison between baseline and each follow-up visit measurements was performed by employing the paired t test. Subsequently, multiple binary logistic regression analysis was used to study predictors of clinical benefit after BM-MNCs transplantation. Statistical analyses were performed using SPSS 21.0 software (SPSS Inc., Chicago, IL, USA). P<0.05 was considered statistically significant.