Summary
Background: Regenerative therapy by autologous stem cell implantation may benefit patients with traumatic spinal cord (SCI). Aims: The aim of this study was to analyze the safety and efficacy of autologous stem cell implantation for traumatic SCI and BI in a clinical setting. Patients and Methods: Eighty one patients (74 with SCI), age between 18 and 65 (median 43), were included in the study. Fifteen to 40×106 CD34+ cells per patient were implanted.
The implantation of the stem cells followed routine neurosurgery procedures. Results: Implantation of the stem cells was tolerated very well and no serious side effects were noted. The lack of control and placebo groups hampers the interpretation of the efficacy analysis. There was, however, improvement in the sensory function in 69 of the 74 patients with SCI.
Conclusion: The implantation of autologous stem cells for traumatic SCI is safe and well tolerated. Proper interpretation of the clinical efficacy necessitates a study that includes control and placebo groups.

A Hadjianev¹, C Botev²,4, V Bussarsky¹, K Romansky¹, N Mirchev¹, K Georgiev¹, L Nuchev¹, St Djendov¹, I Iliev¹, I Tonev²,4, I Altankova³, M Genova², V Hrischev²,4, D Kiurktchiev³, V Shivarov², G Oncheva³, I Shopova³, G Georgiev², E Boteva4, S Hristova4, P Slavov4 and M Mincheff ²,4.
1 – Neurosurgery Clinic, “Aleksandrovska” and “St. Ivan Rilski”
University Hospital, Sofia, Bulgaria
2 – National Center for Hematology and Blood Transfusion, Sofia,
Bulgaria
3 – Central Immunology Laboratory, “St. Ivan Rilski” University
Hospital, Sofia, Bulgaria
4 – Procell Medical Center, Sofia, Bulgaria

Introduction

Spinal cord injury (SCI) remains one of the most devastating conditions in medicine, particularily due to the loss of productive life years and the high economic burden it places on our society. There are limited therapeutic options available to reduce the morbidity and mortality related to SCI. However, recent work with stem cells in repairing SCI appears to be promising making this one of the most exciting frontiers in medicine. Possible mechanisms of stem cell activity include participation in neovascularization1,2, trans-differentiation3, and topical release of growth factors, both by stem cells and platelets4,5. In addition, trans-differentiation of circulating monocytes after extravasation towards neural lineage has been recently proposed6. The aim of the present study was to investigate the safety and efficacy of intrathecal or topical application of autologous bone marrow derived stem cells in patients that have suffered spinal cord injuries.

Patients and Methods:

The study was performed at the St. Ivan Rilski University Hospital in Sofia, Bulgaria. All patients signed an informed consent prior to the inclusion in the study. Seventy four patients with posttraumatic spinal cord injuries were included. Patients with total anatomical injuries were excluded from the study. An attempt to include patients with fresh injuries in the study was made. Bone marrow (5-7 ml/kg total body weight) was harvested by aspiration from the posterior or anterior iliac crest after general anesthesia. Stem cells were processed, concentrated in a semi-closed system by centrifugation and/or spontaneous sedimentation with hydroxyethyl starch (HES), and applied within 3 hours following harvest. Samples from the final preparation were sent for microbiological assays and for flow cytometry. Anti-CD3, CD4, CD8, CD16, CD34, CD45, CD45 and CD56 were purchased from Becton-Dickinson. Samples from each stem cell isolation, as well as sera from patients, were frozen and kept at -80°C for further analysis. For stem cell application, all patients were admitted in the Neurosurgery Clinic of the St. Ivan Rilski University Hospital in Sofia. Mean hospital stay for surgery and stem cell application was 5 days. Preliminary examination of the patients involved laboratory (CBC, blood biochemistry, coagulation status) and neurological testing. Detailed examination of SCI by CT scan, NMR, electromyography, sensory evoked potentials, ASIA scale, neurological and psychological status, as well as self evaluation following a questionnaire were performed prior to and 6-8 months post surgery and stem cell application. Standard neurosurgical procedures such as laminectomy (35 patients; picture 1.1) and interlaminotomy (39 patients) were performed. The type of surgical intervention was discussed for each individual case prior to surgery. Laminectomy was combined with direct implantation of the stem cells at the injury site (picture 1.4). Five to seven injections at both borders of the injury site with an atraumatic needle (31G) totaling 0.1 ml stem cell suspension were performed. Alternatively, interlaminotomy was combined with either intrathecal infusion of the stem cells, or subpial (intraramedullary) injection of the stem cell so that a depot of cells was formed at the injection site. The residual red cells and plasma after bone marrow processing, together with any residual stem cells, were transfused intravenously to the patient.

Results

1. Patients

Seventy four patients with SCI, 33 women and 41 men, age between 18 and 65 (median 43) were included in the study (Table 1).

The causes of the spinal injury were either car accidents 40 (54%), falls 21 (28%), pressed 4 (5.5%), gunshots 8 (11%) or sports activity 1(1.5%). Sixteen percents of the patients had SCIs at the cervical region, 34% at the thoracic region, 34% at the lumbal region and 16% had injuries that extended at several regions (cervico-thoracic, thoraco-lumbar or lumbar-sacral).

2. Stem cell characteristics

Bone marrow (396 +/- 41.6 ml) was aspirated in 50 ml ACD with heparin.

The stem cells were resuspended in autologous plasma (total volume 7-12 ml). The mean number of cells for local application was 4,84 х109 and the total number of cells for local application was always kept under 5×109. The rest of the leukocytes were returned intravenously to the patient together with the residual red cells. Most of the patients underwent one procedure; however, some patients who benefited from the first one had a second one done. The local and/or intrathecal stem cell applications were tolerated well. Common side effects following stem cell application included meningism that developed immediately following the procedure and lasted for about 2 to 3 days, in very few of the patients it lasted 7 to 8 days. Almost all patients experienced fever (37.5°C to 38.5°C) that also lasted 2 to 3 days. In one case the stem cell preparation tested positive for Enterococcus, however, the application, which was performed before the microbiology result became evident, went uneventfully. No further aggravation of the existing neurological symptoms occurred following the procedures. Patients were recruited for neurological examination at day 90 following the procedure. The evaluation of the patients was performed according the ASIA scale. Sixty nine of the patients (93.2%) had improvement in the sensory function, while no improvement was noted in 5 patients. The improvement was noted in patients who had either laminectomy (34 of the 35 patients) or interlaminotomy (35 of the 39 patients). Good action potentials were registered on electromyography but these did not correlate with expected response. The same problem was noted when somatosensory evoked potentials (SSEPs) were tested. Few of the patients, mostly those with existing motor function (14.8%), had improvement in movements. These improvements were registered as an increase of the existing voluntary movements (abduction, adduction, flexion and extension) or new activities in certain muscular groups that were lost after the injury. Control EMGs registered good action potentials which did not always correlate with voluntary muscle function. In some of the patients (9.5%), an absolute increase in the muscle mass of the lower extremities (~1-1.5 cm in circumference) was registered. Three of the patients who had partial sphincter control following the accident regained full control after the procedure. Two of the male patients regained erection. In one patient, the symptoms of insipid diabetes disappeared completely (1.3%). Significant differences in the follow up image results (CT, MRI), were found only in the patients with bone decompression and reduced volume of the posttraumatic hydromyelin cysts (picture 1.3). The rest of the patients had no changes in CT and MRI images before and after stem cell application. Among the patients with improvement following the therapy were such whose injury predated with 10 years the stem cell implantation; one of the best responders was treated 25 years after the trauma.

Discussion

The use of bone marrow derived stem cells for patients with spinal cord injuries is a possible option for treatment, being safe and with acceptable adverse reactions. Our observation is that the clinical response to the treatment depends mainly on the degree of spinal cord injury. The improvement in patients treated more than one year after the accident, included in this study, shows that the use of stem cells for spinal cord injury is possible even years after the trauma. It is not possible from the group of our patients to define, if the way of application of the bone marrow derived stem cells, has influence on the results of the treatment, because in both groups (with laminectomy or interlaminotomy) the results are similar. The improvement in the general condition of the patients, with or without neurological changes, shows that the intravenous application of stem cells also has positive influence on these patients. The second and following procedures are less potent, which requires further evaluation of the necessity and the benefits from more than one procedure. Proper interpretation of the clinical efficacy necessitates a study that includes a control and/or a placebo groups.

Conclusions

1.The application of bone marrow derived leukocyte suspension, including stem cells is a safe medical procedure with acceptable adverse reactions.
2.Improvement in the sensory function occurs in almost all of the patients.
3.Improvement was recorded even inpatients with lumbar infusion of the stem cells, suggesting direct effect of stem cell implantation on sensory improvement.
4.Very minor changes in motor function were noted in several of the patients.
5.Improvement initiated around the 90th day following implantation of the stem cells and fully developed after the 8th month from the procedure.
6.Repetition of the stem cell implantation had a lesser effect if any.

References:

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