Autologous Transplantation of Bone Marrow Mesenchymal Stem Cells on Diabetic Foot

Summary

Objective:

To study the therapeutic effect and safety of transplantation of autologous bone marrow mesenchymal stem cells after amplification on diabetic foot.

Methods:

Type 2 diabetic patients with diabetic foot were enrolled and randomized to either transplanted group or control group. Patients in both groups received the same conventional treatment. Meanwhile, 20 ml bone marrow from each transplanted patient was collected, and the mesenchymal stem cells were separated by density gradient centrifugation and cultured in the medium with autologous serum (from each patient of the transplant group, about 200 ml of whole blood was drained into blood bags, clotted and centrifuged. After that the serum was collected and filtered through a 0.2-μm membrane.) After culture in vitro, mesenchymal stem cells should go through safety evaluation which include culture and check of pathogenic microorganisms (bacteria, mycoplasma, chlamydia, eumycete and viruses) of the cells medium and karyotype analysis of the mesenchymal stem cells. Only the safe cells were harvested and transplanted by multiple intramuscular and hypodermic injections into the impaired lower limbs. Follow-up index include: efficacy (pain,ulcer healing rate, lower limb amputation rate and ,ankle-brachial index,magnetic resonance angiography,electromyogram) and safety (infection of the injection site, immunological rejection, and tumour generation).

Description

RESEARCH DESIGN AND METHODS Diabetic patients with foot ulcers, but without malignant tumor or gangrene above the ankle and/or severe coronary, cerebral,and renal vascular disease, were eligible for participation in this trial. Volunteers who were eligible for this trial were admitted to our hospital from October 2009 to August 2010 and were enrolled and randomized (1:1) to either the transplant group or the control group. This clinical study was approved by the ethical committee board of Southwest Hospital，Third Military Medical University. All participants received written informed consent.

Diabetic foot definitions The definition of Diabetic foot was defined as ankle-brachial index (ABI) ＜0.90 with foot ulcers and abnormality of electromyogram. The Fontaine classification stratified patients as class III (rest pain) or class IV (ulceration and/or gangrene).

bone marrow mesenchymal stem cells（BMMSCs）preparation Bone marrow (20ml) was obtained from the iliac crest of the patients in transplant group after informed consent. The aspirate was diluted 1:2 in Dulbecco's modified Eagle's medium (DMEM)/ F12 (Gibco, Paisley, U.K., http://www.invitrogen.com). After density-gradient centrifugation at 1800g for 20 minutes, the mononuclear cell layer was removed from the interface（the blood plasma layer and red cell layer was collected in order to prepare autologous serum （AS））, washed twice, and suspended in DMEM/F12 at 107 cells per ml，seeded in 50ml culture flask and incubated in 5% CO2 / 95% air at 37oC.Each flask contained DMEM/F12 medium supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 15% FBS from Gibco. On day 1, nonadherent cells were discarded and adherent cells were washed with phosphate-buffered saline (PBS) (Gibco) and then cultured in DMEM/F12 medium with antibiotics and 15% of the same serum. Subsequently, medium containing 15% FBS used for that flask was replaced every 2 days. At approximately 80% confluence, the cells were suspended using trypsin-EDTA and replated at approximately 4,000 cells per cm2. After third passage, in order to reduce 99.99％ heterologous protein（6），the medium containing 15% FBS was removed and the medium containing 15% AS was used for further cell cultures for a week. At the end of the fourth passage，Viable cells were collected and washed thrice by physiological saline.Finally，these cells were diluted by 20ml physiological saline（before the collected 5 day and 3 day，culture of pathogen and karyotype analysis were performed respectively.）. 1 ml of this cell suspension was obtained for Flow Cytometric Analysis and counting.

Preparation of Human Autologous Serum From each patients in transplant group, 30~40ml of the blood plasma layer and red cell layer was transferred to 10-ml tubes with coagulant,and allowed to clot for 4 hours at 4°C to 8°C. Subsequently, the blood was centrifuged at 1800g at 4°C for 15 minutes. Serum was collected and filtered through a 0.2-μm membrane (Sarstedt,Nümbrecht, Germany, http://www.sarstedt.com). Aliquots of the sterile AS were stored at -20°C.

Flow Cytometric Analysis For flow cytometric analyses of surface molecule expression, the following Mabs directly conjugated to fluorochromes were used:CD34-fluorescein isothiocyanate (FITC)，CD44-phycoerythrin(PE)，CD105-PE (BD Biosciences, San Diego, http://www.bdbiosciences.com). Irrelevant control Mabs were included for all fluorochromes.Cells were coated with directly conjugated Mab at room temperature for 15 minutes, washed, and fixed in 1% paraformaldehyde. Cells were analyzed using a FACSCalibur flow cytometer (Becton, Dickinson and Company,San Jose, CA, http://www.bd.com). Gates were set based on staining with combinations of relevant Mab and irrelevant Mab so that no more than 5% of the cells were positive using irrelevant Mab.

Examination of cells'Safety MSC chromosome preparation and cytogenetic analysis： Chromosome samples were prepared according to the method described previously (E Z, 1997) and the karyotype was analyzed by G-banding techniques.For each individual patient, 8～15 metaphase cells were analyzed usinga LUCIA chromosome automatic analysis system.

Cultivation of pathogenic microorganism： the medium in the culture flask were cultured in order to find pathogen，which included bacterium、mycoplasma、chlamydia、eumycete、virus.

Administration of therapy All of the patients received conventional care for their ulcers，which included adjustment of blood glucose、blood pressure and blood fat，debridement in order to remove extensive callus and necrotic tissue.，pressure-relief after wound dressing, application of antibiotics（Broad spectrum antibiotics were prescribed if ulcers showed clinical signs of infection. Adjustments to the treatment were performed when indicated on the basis of microbiologic cultures and sensitivity testing）. The patients in control group only received conventional care.In the transplant group, besides the conventional care the patients received the transplantation of his/her own bone marrow mesenchymal stem cells（BMMSCs）.

Autologous transplantation of bone marrow mesenchymal stem cells（BMMSCs） Under the Strictly aseptic conditions，intramuscular injection of Crispin melanate 100mg was performed at rump for relieving pain. After 30 minute, diseased lower limb was intramuscularly injected (20 sites, 3×3 cm distance,1~1.5 cm deep, 0.5～1ml BMMSCs per site) into leg . The basilar part of each foot ulcer and subcutaneous tissue around it also were injected about 2ml BMMSCs .

observation index and assessment guidelines Clinical data, medication, and safety laboratory data were prospectively collected, and follow-up visits were performed 3 weeks before and 3 months after transplantation. Specific attention was paid to any potential adverse effects specifically because of transplantation during follow-up.The criteria of Tateishi-Yuyama (15) and Oyibo (16)were used to assess limb status. Rest pains on rating scales ranged from 0 points for the best (complete relief of pain with no use of analgesics) to 4 points for the worst result. Ulcer healing rate（closure patient/ total patient in a group）of both control and transplant group were calculated at every week respectively after transplantation. ABI was measured for all patients before and after treatment by laser Doppler (Lisca Developments, Linko ping, England) in a room adjusted for 21°C.Electromyogram was measured for all patients before and after treatment.

analysis of nuclear magnetic resonance image of lower limbs blood vessel The patients were subjected to analysis of nuclear magnetic resonance image（MRI）of lower limbs blood vessel 1 week before and 8 weeks after transplantation. The scores of blood vessel image (11) for the formation of new collateral vessels were assessed as+0 (no collateral development),+1 (slight), +2 (moderate), and +3(rich).

Statistical analysis Continuous variables are presented as means ±SD. Changes in variables from baseline to week 8 were analyzed by the paired or two-tailed Student's t and X2 tests. X2 Analysis with likelihood ratio was performed. Statistical significance was assumed at a value of P＜0.05. All statistical analysis was performed with SPSS version 10.1 for Windows (SPSS,Chicago, IL).

Study Design

Allocation: Randomized, Control: Placebo Control, Endpoint Classification: Safety/Efficacy Study, Intervention Model: Parallel Assignment, Masking: Double Blind (Subject, Caregiver, Investigator, Outcomes Assessor), Primary Purpose: Treatment

Conditions

Autologous Transplantation

Intervention

Autologous transplantation of mesenchymal stem cells

Location

the south west Hospital
Chongqing
Chongqing
China
400038

Status

Recruiting

Source

Third Military Medical University

Results (where available)

View Results

Links

• Source: http://clinicaltrials.gov/show/NCT00955669
• Information obtained from ClinicalTrials.gov on July 15, 2010

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