Progressive Multiple Sclerosis and Pressure Ulcer Successfully Treated for the

First Time with Autologous Mesenchymal Stem Cells

 A Case Report

Authors: Mirela Patricia Sirbu Boeti, Adriana Dulamea, Coralia Bleotu, Catalin Efrimescu, Mihaela Chivu, Lucia Moldovan, Dan Lucian Dumitru, Elena Ciucur, Denisa Dragu,

Irinel Popescu

Introduction

Successful clinical treatments with stem cells of patients with multiple sclerosis (MS) have been sporadically reported. Patients with paraplegia secondary to MS are at risk to develop pressure ulcers. Mesenchymal stem cells (MSCs) were observed to facilitate the healing of severe burns or difficult leg ulcers.

We present the case of a young paraplegic patient with MS that received local application of autologous mesenchymal stem cells with the first intent to accelerate the healing of two refractory leg pressure ulcers. Considering the autoimmune etiology of MS and the immunomodulatory functions of MSCs, a possible beneficial effect for the background disease was anticipated and previously discussed with the patient.

The patient presented was treated at Fundeni Clinical Institute, Carol Davila University of Medicine and Pharmacy.

Case presentation/Background

On March 2003 a 46-year-old man patient was admitted at Department of Neurology, Fundeni Clinical Institute for paresthesia and pain in both legs and progressive paraparesis (left leg 2/5, right leg 3/5). Cerebral MRI showed 3 small demyelinating lesions with hyperintensity in T2 localized in the subcortical zone of the left frontal and right pariental lobe. Spinal cord MRI showed spinal lesion with hyperintensity in T2, FLAIR and hypointensity in T1 at the level of T2-T3 vertebrae corresponding to a demyelinating lesion. Based on the clinical presentation and MRI, the possible diagnosis was multiple sclerosis – spinal form or myelitis with Expanded Disability Status Scale (EDSS) 6.5. Oral corticotherapy was instituted and followed by partial recovery, the patient becoming able to ambulate with constant assistance and EDSS improving to 6.

On September 2006, pressure ulcers of left hallux and right foot occurred and were successfully treated by conventional methods at another hospital.

On May 2007, readmission of the patient was imposed for the onset of optic neuritis of right eye, bilateral hypoacusia (right > left), paraplegia, bilateral pyramidal syndrome, hypoesthesia in both legs, bilateral L5 radiculalgia, urinary retention, and fatigue. On MRI it was observed no demyelinating lesion on cervical-thoraco-lumbar spinal cord. The electromyogram was also normal. The patient was diagnosed with primary progressive multiple sclerosis.

Treatment with Solu-Medrol 1 g/day i.v. was administered for 5 days, and then changed to oral Medrol 32 mg/day gradually tapered until cessation and cyclophosphamid 200 mg/day i.v. for 5 days. After receiving Solu-Medrol i.v. patient was ambulatory without aid (EDSS 5.5), but 2 months later, after withdrawal of oral Solu-Medrol, he became restricted to bed (EDSS 8). Oral Solu-Medrol 8 mg/day was resumed as chronic oral treatment.

On February 2008, the patient developed pressure ulcers of the left lateral malleolus and thigh.

On April 2008, the patient was readmitted to the Department of Neurology – Fundeni Clinical Institute, presenting bilateral optic neuritis, bilateral hypoacusia, diplopia for central, right and left gaze, right facial palsy, tetraparesis (paraplegia 0/5 and diparesis 3/5), anesthesia of left inferior limb, urinary retention, fatigue, anxiety, severe chronic ulcers of the left lateral malleola and thigh with important trophic changes, absent deep tendon reflex (DTR) of left arm and leg, exaggerated DTR of right arm and leg, and being unable to move in bed by himself.

For the ulcer pressures, he was referred at the Department of Surgery, Fundeni Clinical Institute. Two infected stage IV pressure ulcers were noticed on the lateral left malleola (8/7 cm) and respectively on lateral side of tight (3 cm). Cultures of wound secretions came positive for Proteus and Stafilococcus aureus. A third stage III ulcer pressure was observed on right calcaneum. Cerebral and spinal MRI were both normal as was the electromyogram. Cerebrospinal fluid had normal albumin, no cells, and normal glucose. The progression of primary progressive multiple sclerosis was certain with EDSS 9. Oral Medrol 8 mg every other day was started. Systemic polyantibiotherapy and local treatment with Sorbalgon, AtraumanAg, and TenderWet were conducted to clean the wounds.

At the end of April 2008 the informed consent and approval by the Ethics Committee at Fundeni Clinical Hospital were obtained for application of biological dressing with autologous mesenchymal stem cells.

Preparation of biological dressing with Mesenchymal Stem Cells MSCs

The patient was offered a plastic surgery consult and the possibility of the treatment of bed sores with autologous skin grafts. He consented to receive the treatment of pressure ulcers with autologous MSCs. The time of in vitro cell propagation was beneficial to prepare the wound beds for biological dressing.

The patient was investigated to exclude any ongoing non-occult malignant neoplasm. The viral status usually checked for transplanted patients was also obtained and came negative.

The bone marrow aspirate was obtained from the right posterior superior iliac spina. MSCs were isolated from patient’s bone marrow aspirate. Mononuclear cells were separated by centrifugation over a Biocoll gradient (Biochrom, Germany) and suspended in alpha-MEM containing 10% autologous serum and penicillin-streptomycin mixture (Biochrom, Germany), followed by plating at an initial seeding density of 1 x 10⁶/cm². After 3 days, the nonadherent cells were removed and monolayers of adherent cells were cultured until they reached confluence. Cells were trypsinized with 0.25% trypsin and subcultured at densities of 5000-6000 cells/cm². After the first passage, the culture of MSCs became homogenous and demonstrated a high proliferation potential. The cell surface marker identification was performed by Epics XL FACS analyzer (Beckman Coulter) using fluorescein isothiocyanate (FITC) or phycoerythin (PE)-labeled monoclonal antibodies. Phenotypic characterization confirmed MSCs: CD105+, CD90+, CD34-, CD45-, CD3-, CD14-. A normal kariotype of MSCs was confirmed. Biological dressing was made of 8 x 10⁶ MSCs seeded into a 30 cm² collagen-agarose sponge placed on a decellularized human arterial fragment. The collagen-agarose sponge and decellularized human arterial fragment were obtained using the same protocol previously described by us.

Stage IV pressure ulcers were completely covered with the biological dressing containing MSCs.

Follow-up and progression of case

Healing of the wounds was followed on a daily base for the first two weeks and then every two days for other two weeks. Two months later the left lateral femoral pressure ulcer was completely epithelized with 2 mm central depression. The healing of the left malleolar wound was completed five months later with no depression nor impediment in ankle joint movement. The right calcanean bed sore healed with dry dressing.

The neurological improvement was daily recorded by the neurologist. The neurologic impact of transplanted MSCs was heralded by the recovery of sensitivity at the treated leg. At the neurological clinical assessment done one week later diplopia and facial palsy were absent.  At two weeks patient experienced hypoesthesia of the left leg, improved strength in both arms, movements of the fingers of both legs, and one month later patient recovered his sensibility for both legs, was able to move in bed by himself, sit, and move inferior limbs against gravity. Paraparesis was 3/5 for left leg and 4/5 for right leg and the spasticity of both legs was noted being greater for the left one and EDSS improved from 9 to 8. At 2 months after autologous cell therapy the patient was able to walk 10 m with constant bilateral assistance and EDSS became 7. At 4 months he proved touch and pain hypoesthesia, paraparesis (left leg 3/5, right leg 4/5), spasticity on flexors of the left limb, no arm deficits, no ataxia, being able to walk 40 m with constant bilateral assistance with EDSS 6.5. At two years after MSC transplantation the patient rides bicycle and is able to shovel snow, being free of medication.

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Discussions

The successful healing of both pressure ulcers was expected, however the patient had significant neurological improvement.

Our patient had an unequivocal progressive motor decline unresponsive to corticosteroid and cytotoxic therapy.

The unique therapeutic results rise essential questions:

1)    What is the mechanism of action of MSCs in the treatment of MS: plasticity with the possibility of differentiation in neurons or glial cells (e.g. oligodentrocytes), facilitation of regeneration of nervous tissue by cytokines secreted by MSCs, immunomodulation?

2)    Is it possible to quantify the predicted effects of MSCs pre- and postengraftment?

3)    What is the best source of MSCs?

4)    What is the number of MSCs needed to stop and reverse the neurological lesions in MS?

5)    What is the best administration route of MSCs for the treatment of MS?

6)    What is the impact of MSCs on the long-course of MS?

7)    Are MSCs safe with regard to their malignancy potential or potential of differentiation in unwanted cells?

8)    What is the best protocol of in vitro expansion of MSCs?

Treating a very debilitated patient, we aimed for an autologous graft in order to alleviate the side effects of an allograft. The bone marrow aspiration is a facile source of MSCs. Taken from our patient it provided a sufficient number of MSCs to start with. We aimed to restrict the number of culture passages to minimize the risk of in vitro mutations. In order to avoid the expansion of already mutated MSCs, cell kariotype was checked. However the risk of preexisting small mutations could not be totally excluded. As an important issue, we avoided the use of fetal calf serum for cell culture. Instead we used the patient’s plasma to maintain MSC culture. The administration of these cells was facilitated by the presence of bed sores. Not only that the migratory capacity of these cells but also their “clever” target toward previously injured tissues was already proved by other authors. Simple application of MSCs embedded in a collagen-agarose sponge had the advantage of alleviating the cell damage possible associated with injection barotrauma.

The MSCs applied on bed sores alleviated the neurologic symptoms of a patient with MS removing the risks and avoiding the side effects secondary to other administration routes (e.g. systemic intravenous or intrathecal route).

After two years of free symptom period, our patient with progressive MS needs no medication. At the site of bed sores there are well-healed scars. Although the patient has no present complaint, he will be kept for follow-up for further periodical neurological assessments.

Conclusion

Local application of autologous in vitro expanded MSCs within biological dressing accelerated the healing of stage IV pressure ulcers and completely reversed the neurologic manifestations in a young patient with primary progressive multiple sclerosis. After two years the patient is free of medication with no health complain.

last updated 2010-03-17