Abstract

Olfactory ensheathing cells transplanted into the injured spinal cord in animals promote regeneration and remyelination of descending motor pathways through the site of injury and the return of motor functions. In a single-blind, Phase I clinical trial, we aimed to test the feasibility and safety of transplantation of autologous olfactory ensheathing cells into the injured spinal cord in human paraplegia. Participants were three male paraplegics, 18–55 years of age, with stable, complete thoracic injuries 6–32 months previously, with stable spinal column, no implanted prostheses, and no syrinx. Olfactory ensheathing cells were grown and purified in vitro from nasal biopsies and injected into the region of damaged spinal cord. The trial design includes a matched injury group as a control for the assessors, who are blind to treatment status. Assessments, made before transplantation and at regular intervals subsequently, include MRI, medical, neurological and psychosocial assessments, and standard American Spinal Injury Association and Functional Independence Measure assessments. One year after cell implantation, there were no medical, surgical or other complications to indicate that the procedure is unsafe. There is no evidence of spinal cord damage nor of cyst, syrinx or tumour formation. There was no neuropathic pain reported by the participants, no change in psychosocial status and no evidence of deterioration in neurological status. Participants will be followed for 3 years to confirm long-term safety and to compare neurological, functional and psychosocial outcomes with the control group. We conclude transplantation of autologous olfactory ensheathing cells into the injured spinal cord is feasible and is safe up to one year post-implantation.

Introduction

There are no effective and accepted treatments that can repair the neurological damage in human spinal cord injury but there are numerous reports of successful approaches in animal models of spinal cord injury, including transplantation of olfactory ensheathing cells.

Olfactory ensheathing cells are specialised glial cells that surround the olfactory sensory axons in the nose. They have properties of Schwann cells in promoting and assisting growth of axons (Doucette, 1995). They are unique among the glia in residing both inside and outside the central nervous system, in the olfactory bulb and olfactory nerve, respectively (Gudino-Cabrera and Nieto-Sampedro, 2000). These properties have led to an increasing use of olfactory ensheathing cells in preclinical models of transplantation for spinal cord repair including complete transection, hemisection, tract lesion, and contusion with over 50 studies published in the last 10 years (Ramon-Cueto and Nieto-Sampedro, 1994; Franklin et al., 1996; Smale et al., 1996; Li et al., 1997; Franklin and Barnett, 2000; Raisman, 2001; Barnett and Chang, 2004).

Transplantation of olfactory ensheathing cells into the lesioned corticospinal tract led to recovery of paw usage (Li et al., 1997), transplantation after complete transection of the spinal cord led to recovery of coordinated walking (Ramon-Cueto et al., 2000) and transplantation after spinal cord hemisection led to recovery of paw use and climbing (Keyvan-Fouladi et al., 2003; Li et al., 2003). Recently olfactory ensheathing cell transplants were reported to assist recovery after spinal cord injury of bladder function (Pascual et al., 2002) and phrenic nerve activity (Li et al., 2003; Polentes et al., 2004). Olfactory ensheathing cells do not myelinate the olfactory nerve but when transplanted into the spinal cord they help to myelinate the regrowing axons in the region of injury after corticospinal tract lesions (Li et al., 1998) and they support remyelination of axons in several demyelinating models (Franklin et al., 1996; Imaizumi et al., 1998; Kato et al., 2000; Radtke et al., 2004).

Biopsy of the olfactory mucosa provides an accessible source of olfactory tissue in adult humans (Féron et al., 1998) from which olfactory ensheathing cells can be grown (Bianco et al., 2004). Nasal olfactory ensheathing cell transplants assist recovery after spinal cord injury, including complete transection (Lu et al., 2001; Ramer et al., 2004) and there is evidence that adult olfactory tissue is effective when transplanted 1 month after spinal cord transection in the rat (Lu et al., 2002).

We are doing a single-blinded, controlled trial to establish the safety and feasibility of intraspinal transplantation of autologous olfactory ensheathing cells in human spinal cord injury. This is a report of the outcome of the trial 1 year after transplantation.

Methods

Participants

Participants were eligible if they were 18 years or older, had a complete spinal cord injury (American Spinal Injury Association Category A, ‘ASIA A’) between T4 and T10 of traumatic aetiology with no evidence of ongoing recovery of spinal cord function and were between 6 months and 3 years post-injury (i.e. they were neurologically stable). Exclusion criteria included: substance abuse, syringomyelia, spinal vertebral instability, major concurrent medical illness (e.g. carcinoma, auto-immune disease, diabetes mellitus) and ASIA Impairment Scale category other than A. Subjects with major and current psychiatric illness, who had significant traumatic brain injury associated with the spinal cord injury or who were otherwise considered unable to provide fully informed consent were also excluded. Selected participants were thoroughly informed of the trial and its attendant risks and asked to consent to be in one of two groups, those receiving cell transplants or the control group.

The trial was approved by the ethics committees of the Princess Alexandra Hospital and of Griffith University according to guidelines of the National Health and Medical Research Council of Australia. The trial was approved by the Therapeutic Goods Administration of Australia and accordingly monitored by the responsible ethics committees.

Nasal biopsy and olfactory ensheathing cell culture

After formal admission to the trial, the three participants in the transplant group underwent general anaesthesia for a nasal biopsy to produce olfactory ensheathing cells. Biopsies were taken from the nasal septum in the superior region of the nasal cavity close to the cribriform plate, under visual guidance via a nasal endoscope. Three medium-sized (∼5 mm2) or two large-sized (∼10 mm2) biopsies of olfactory mucosa were collected via one naris of each patient. The tissue was excised using an ethmoid forceps (Richard Wolf Medical, number 8211.511; Hoyland Medical, Camp Hill, Queensland) and placed immediately into a sterile container in cold culture medium and all subsequent processing was done under aseptic conditions. The culture medium was DMEM/HAM F12 containing 10% foetal calf serum, penicillin/streptomycin/fungizone (100 U/ml, 100 and 2.5 µg/ml respectively, Invitrogen). This was transported on ice from the operating theatre to the cell culture facility where the olfactory mucosa was allowed to rest for 2 h. Then the tissue was incubated for 45 min at 37°C in 2 ml of a 2.4 U/ml dispase II solution (Boehringer).

Laminae propriae were carefully separated from the overlying epithelium under a dissection microscope using a microspatula and cut into pieces of ∼0.05 mm2 using a McIlwain tissue chopper (Brinkmann) (Féron et al., 1998; Bianco et al., 2004). The minced lamina propria was incubated at 37°C in collagenase H (0.3%; Sigma) for 15 min and mechanically triturated every 5 min using a flame polished Pasteur pipette. The enzymatic activity was stopped using a 0.5 mM EDTA solution (Invitrogen) and the dissociated cells were pelleted before being plated onto poly-l-lysine (1 g/cm2, Sigma) pre-treated dishes and grown at 37°C in 95% O2/5% CO2. Forty eight hours after the initial plating, the serum-containing medium was removed and cells were fed with DMEM/HAM F12 supplemented with penicillin/steptomycin, insulin (10 µg/ml, Invitrogen), selenium (6.7 µg/ml, Invitrogen), transferrin (5.5 µg/ml, Invitrogen) and recombinant human neurotrophin 3 (NT3, 25 ng/ml, Alomone). This medium was replaced every 3 days and the cells were passaged at intervals when they reached confluency and plated into 75 cm2 flasks with the procedure repeated over 4 weeks to build up an adequate supply of cells. On the day of transplantation the cells were harvested by trypsinisation after which the enzymatic activity was stopped by soybean trypsin inhibitor (Invitrogen). Cells were pelleted (300 g), transferred into sterile 200 µl PCR tubes at a concentration of 2 million cells/25 µl of serum-free medium, and delivered to the operating theatre.

Cell culture quality control

Seven to fourteen days prior to transplantation the olfactory ensheathing cells were checked immunologically for their phenotype. A sample of cells was transferred onto a glass 8-well Labtek chamber slide (Nunc), grown for 48 h, fixed for 10 min in methanol at −20°C, and immunostained with antibodies raised against GFAP (glial fibrillary acidic protein, polyclonal, 1/500, DAKO), S100 (polyclonal, 1/500, DAKO), primate p75NTR (the low affinity neurotrophin receptor, monoclonal, un-purified antibody produced by an hybridoma from ATCC) and HNK1 (R-phycoerythrin-conjugated polyclonal, 1/200, Sigma). Primary antibodies were labelled with a fluorescent secondary antibody, except for anti-HNK1 which was fluorescent. Cells were examined and quantified microscopically and photographed digitally.

Five to nine days prior to transplantation, samples of the culture medium were tested for sterility. From each culture flask, 0.5 ml of culture medium was transferred into a bottle containing 70 ml of Brewers Broth. The inoculated broth was then returned to the Microbiology Department, Queensland Health Pathology Service, Princess Alexandra Hospital for processing. Brewers Broth was chosen as a universal culture medium for sterility testing, and the large volume used was in order to dilute the antibiotics from the original cell culture medium. The presence of bacteria or fungi in a sample would render that flask unusable and it would be discarded. After each transplantation, several aliquots of remaining cells were retained, assessed for viability, and grown as described above to assay for the development of bacterial or fungal infection that might have been introduced by the transfer procedures. None was found prior to tissue collection before transplantation. Blood was drawn from the cell culturist (F.F.) and assessed for viral infection. None was found.

Injection device and filling station

An injection system was designed to allow transplantation of the cells via microinjection and the re-filling of the injection syringes within the aseptic surgical field during the transplant operation (Fig. 1). The injection device was based on the HLA (Terasaki) Dispenser (Hamilton Company, Catalog Number 83787) (Fig. 2). This six-syringe dispenser was modified to hold a single syringe. The device is operated by moving a spring-loaded lever, whose gearing incrementally advances the syringe plunger. Each lever press advances the syringe plunger a set distance, ejecting 0.55 µl from the syringe. The injector device was modified to attach to the micromanipulator (Fig. 2B and C) and filling station with a twist-operated lock (Fig. 2D and E). The injector device was fitted with a 25 µl Hamilton syringe with 28 ga bevelled needle. The micromanipulator (MM-33, Fine Science Tools, Vancouver, Canada) was modified to attach to the circular rail of the spinal orthopaedic stabilization system (Synthes Synframe) that mounts to the operating table (Fig. 2C). An extension arm on the micromanipulator contained a docking aperture through which the twist-operated lock on the injector device was attached, holding the injector device in parallel with the z-axis of the micromanipulator (Fig. 2). The filling station was designed to hold the injector device so that the syringe needle tip could be lowered with precision into an microcentrifuge tube (Eppendorf) containing prepared cells (Fig. 2D and E). It had a docking port to hold the injector device and a z-axis manipulator to allow the injector device to be raised and lowered. The top of the syringe plunger was locked between two bars to provide firm retraction to draw up the cells. The centrifuge tube fitted into a fixed receptacle on the base of the filling station. Prior to surgery the Hamilton syringes were sterilized by gamma-irradiation and the injection device and filling station were sterilized with ethylene dioxide. Cells (80 000 cells/µl in culture medium) were delivered through a 28 ga needle with a 30° bevel which was shown, by experiment, not to decrease the viability of the cells (Féron, unpublished data).

Fig. 1

Photograph of cell injection system showing syringe (s), injection device (i), and micromanipulator (m) mounted on the circular surgical frame during an operation.

Fig. 1

Photograph of cell injection system showing syringe (s), injection device (i), and micromanipulator (m) mounted on the circular surgical frame during an operation.

Fig. 2

Injector device and filling station. (A) The injector device with syringe attached to the micromanipulator. The injector device (I) holds the syringe in a clamp (C). It is designed around a linear ratchet operated by a dispenser lever (D). Depressing the lever in the direction of the lower arrow, moves the ratchet shaft (R) in the direction of the upper arrow. The shaft is attached to the plunger of the syringe (P) so that movement of the lever ejects a set volume from the tip of the syringe needle (N). The injector device with syringe is attached to the micromanipulator (M) via a twist-lock (T). (B) Micromanipulator in plan view and (C) side view. The micromanipulator is composed of four micrometers (M) controlling movement in three planes (straight arrows). Two micrometers (coarse and fine) control vertical movement. The injector device is locked to the attachment arm (A) through the aperture in the arm (a). The micromanipulator is fixed to the surgical stabilisation system with the vice (V), locked and unlocked by lever (L). Rotation of the manipulator in the horizontal plane (circular arrows) is possible when the vice lock is loosened. Rotation of the manipulator in the vertical plane is also possible when the vice lock is loosed allowing rotation around the attachment bar (b). (D) Filling station in side view with injector device attached and (E) unattached. The injector is fixed to the filling station with the twist-lock (T) through the docking port (d) as shown by the arrow on the right. The device is moved up and down with a ratchet (R) moving the syringe needle into and out of the micro-centrifuge tube of cells (E) located in the holder (H) fixed to the base of the filling station. The syringe plunger is drawn upwards (top arrow in D) until it meets a stopping plate (S) that limits its movement.

Fig. 2

Injector device and filling station. (A) The injector device with syringe attached to the micromanipulator. The injector device (I) holds the syringe in a clamp (C). It is designed around a linear ratchet operated by a dispenser lever (D). Depressing the lever in the direction of the lower arrow, moves the ratchet shaft (R) in the direction of the upper arrow. The shaft is attached to the plunger of the syringe (P) so that movement of the lever ejects a set volume from the tip of the syringe needle (N). The injector device with syringe is attached to the micromanipulator (M) via a twist-lock (T). (B) Micromanipulator in plan view and (C) side view. The micromanipulator is composed of four micrometers (M) controlling movement in three planes (straight arrows). Two micrometers (coarse and fine) control vertical movement. The injector device is locked to the attachment arm (A) through the aperture in the arm (a). The micromanipulator is fixed to the surgical stabilisation system with the vice (V), locked and unlocked by lever (L). Rotation of the manipulator in the horizontal plane (circular arrows) is possible when the vice lock is loosened. Rotation of the manipulator in the vertical plane is also possible when the vice lock is loosed allowing rotation around the attachment bar (b). (D) Filling station in side view with injector device attached and (E) unattached. The injector is fixed to the filling station with the twist-lock (T) through the docking port (d) as shown by the arrow on the right. The device is moved up and down with a ratchet (R) moving the syringe needle into and out of the micro-centrifuge tube of cells (E) located in the holder (H) fixed to the base of the filling station. The syringe plunger is drawn upwards (top arrow in D) until it meets a stopping plate (S) that limits its movement.

Neurosurgical and transplantation procedure

The cell transplantation surgery for the three subjects in the control group took place 4, 5 and 10 weeks respectively following the nasal biopsy.

Surgical technique

If previous surgery had not been performed for management of the primary fracture complex (Patient 1), pre-operative level localization was undertaken with fluoroscopy and methylene blue injection by a radiologist the day prior to surgery. General endotracheal anaesthetic was administered and the patient was placed in prone position supported on a four-post frame. The skin was doubly prepared with alcoholic betadine and the proposed incision line was infiltrated with 0.5% bupivacaine with 1 in 200 000 adrenaline. One gram of intravenous kefazolin (Patients 2 and 3) or Timentin 3.1 g and gentamicin 240 mg (Patient 1) was administered on induction and then repeated at 4 h. Spinal cord monitoring was not used.

A midline incision was made to expose the site of the injury and normal vertebral column rostral and caudal to the injury. Multiple level laminectomy was performed to expose the dura at the site of the injury and rostral and caudal to it. Care was taken not to interfere with the facet joints. A midline durotomy was performed away from the site of injury and the opening completed by splitting the normal dura and sharp dissection, after dissection of adhesions, through the injury.

Using magnification of an operating microscope (NC4, Zeiss Corporation), a dorsal adhesolysis was performed using sharp and blunt dissection methods through the injury to restore as normal anatomy of the cord as possible. The cells were then injected, using the device previously described, into the normal spinal cord adjacent to the injury and into the injury itself. In general, injections were made into the normal cord in a matrix pattern of three rows, 5 mm apart, and five columns, 1 mm apart. At each injection site, 1.1 µl of cell solution was delivered at four depths, 1 mm apart through the antero-posterior substance of the cord (Fig. 3). Systematic injection into the damaged cord was made difficult by its irregular shape and injections were performed using both the frame-assisted and freehand methods. The patients received different numbers of cells: Patient 1, 12 million; Patient 2, 24 million; and Patient 3, 28 million.

Fig. 3

Schematic of injection sites in an injured spinal cord. The injured spinal cord (double arrow) is shown between apparently uninjured spinal cord rostrally and caudally. A five by three grid of injection sites was regularly arrayed rostral and caudal to the injured spinal cord and less regularly than shown through the damaged cord (black dots). Rows were 5 mm apart and columns were 1 mm apart where possible.

Fig. 3

Schematic of injection sites in an injured spinal cord. The injured spinal cord (double arrow) is shown between apparently uninjured spinal cord rostrally and caudally. A five by three grid of injection sites was regularly arrayed rostral and caudal to the injured spinal cord and less regularly than shown through the damaged cord (black dots). Rows were 5 mm apart and columns were 1 mm apart where possible.

The dura was primarily closed with absorbable sutures (Vicryl, Ethicon Johnson and Johnson) and covered with Gelfoam (Ethicon Johnson and Johnson). A wound drain was placed on the Gelfoam and connected to a collection bag to which suction was not applied. The wound was then closed in layers.

Results

From over 600 enquires, 21 persons were suitable for assessment. Of these 9 were excluded and 12 underwent the initial assessment. Of these 3 chose not to participate, 3 were excluded, and 6 were enrolled in the trial, 3 as controls and 3 as transplant recipients. All were males, aged 18–55 years with the period since their injury ranging from 18 to 32 months. All will be assessed regularly for 3 years after enrolling in the trial. The results below pertain only to the safety of the surgical procedure and medical status at 1 year after surgery only for the transplant recipients. A future report will present the results from all assessments for transplants and controls at the end of the 3 year study period.

Olfactory ensheathing cell culture

Olfactory ensheathing cell cultures contained predominantly olfactory ensheathing cells, identified by their immuno-staining for GFAP, S100 and p75NTR (Fig. 4). None of the cells was immuno-positive for HNK-1, a marker for Schwann cells, which can also be positive for GFAP, S100 and p75NTR, confirming our previous observations (Bianco et al., 2004). The percentages of S100-positive and GFAP-positive cells were >95%. The percentage of p75NTR-positive cells ranged from 76 to 88%. In the pre-transplantation testing of the olfactory ensheathing cell cultures, 1 flask (out of 57) grew Candida parapsilosis. This was discarded. After transplantation surgery, the surplus cells were cultured. None of these flasks of cells showed any evidence of contamination after 4 weeks in vitro.

Fig. 4

Immunostaining of olfactory ensheathing cells purified from nasal biopsy of olfactory mucosa. Seven to fourteen days prior to transplantation, an aliquot of cultivated olfactory ensheathing cells from each patient was replated onto a glass 8-well Labtek chamber slide, grown for 48 h and immunostained with antibodies raised against GFAP (A), S100 (B), primate p75NTR (C) or HNK1 (D). Close to 100% of the cells expressed GFAP and S100. The percentage of cells expressing p75NTR ranged from 76 to 88%. No cell expressing HNK1, a marker for Schwann cells, was found.

Fig. 4

Immunostaining of olfactory ensheathing cells purified from nasal biopsy of olfactory mucosa. Seven to fourteen days prior to transplantation, an aliquot of cultivated olfactory ensheathing cells from each patient was replated onto a glass 8-well Labtek chamber slide, grown for 48 h and immunostained with antibodies raised against GFAP (A), S100 (B), primate p75NTR (C) or HNK1 (D). Close to 100% of the cells expressed GFAP and S100. The percentage of cells expressing p75NTR ranged from 76 to 88%. No cell expressing HNK1, a marker for Schwann cells, was found.

Surgical procedures

Findings at surgery

The vertebral level of injury, laminectomy and estimated number of cells injected are presented in Table 1. In all three patients, the surgery was in the thoracic spine, and consisted of multiple level laminectomies. The dura was adherent to the posterior elements of the spine in two cases. In Patient 2, the dura was adherent to the bone at the apex of the kyphotic deformity by scar tissue. In Patient 3, the dura was adherent at the edges of the previous laminectomy defect.

Table 1

Surgical details


 
Previous surgery
 
Length of cord injury (mm)
 
Number of injections
 
Estimated number of cells transplanted (million)
 
Patient 1 Nil 25 270 12 
Patient 2 Pedicle screw instrumentation at time of injury. Subsequently removed. No laminectomy 74 545 24 
Patient 3 Pedicle screw instrumentation at the time of injury. Subsequently removed. Laminectomy at time of original surgery 21 630 28 

 
Previous surgery
 
Length of cord injury (mm)
 
Number of injections
 
Estimated number of cells transplanted (million)
 
Patient 1 Nil 25 270 12 
Patient 2 Pedicle screw instrumentation at time of injury. Subsequently removed. No laminectomy 74 545 24 
Patient 3 Pedicle screw instrumentation at the time of injury. Subsequently removed. Laminectomy at time of original surgery 21 630 28 

In all three cases, the spinal cord was in continuity although it was composed of a cystic area at the site of injury which was encapsulated by tissue presumed to be pia. The pia was in turn tethered to the arachnoid with scar tissue. During the adhesolysis, the cyst cavity was entered in one patient with egress of some fluid. In all three cases, the cavity was smaller but remained present after adhesolysis. The spinal cord exhibited a normal macroscopic appearance adjacent to the area of injury in all cases. In Patient 3, blood vessels traversed the surface of the area of injury, a feature not seen in the other patients.

Perioperative complications

There was no evidence of local infection at the site of surgery in any of the patients and no evidence of any serious infective process related to the surgery, or the transplantation procedure.

One year post-transplantation

No patients experienced infection, leakage of cerebrospinal fluid or progressive spinal deformity. There was no deterioration in neurological or functional level in any of the participants. There was no deterioration in sensory or motor function. There was also no worsening of respiratory function. None of the patients experienced any new or additional neuropathic pain. There was no worsening in the severity of spasticity in any participants. There was no deterioration in psychosocial status and all participants appeared to be coping well. None of the participants had required any additional or unplanned counselling regarding the trial.

Radiology

Because of the limitations of current 1.5 T MR imaging, an increase in volume of the spinal cord would be difficult to define until it were quite extensive. Given this limitation, there appears to be no significant change in the mass of the cord at the injury level in the 12 month scans (Figs 57).

Fig. 5

Sagittal MR imaging of Patient 1 taken before olfactory ensheathing cell transplantation (top pair) and 12 months afterwards (bottom pair). On the left are T1 weighted images with gadolinium contrast. On the right are matched T2 weighted images. The pre-operative images show an intact vertebral column and a short segment spinal cord injury. The 12 month post-operative images demonstrate no significant changes apart from the laminectomy.

Fig. 5

Sagittal MR imaging of Patient 1 taken before olfactory ensheathing cell transplantation (top pair) and 12 months afterwards (bottom pair). On the left are T1 weighted images with gadolinium contrast. On the right are matched T2 weighted images. The pre-operative images show an intact vertebral column and a short segment spinal cord injury. The 12 month post-operative images demonstrate no significant changes apart from the laminectomy.

Fig. 6

Sagittal MR imaging in Patient 2 taken before olfactory ensheathing cell transplantation (top pair) and 12 months afterwards (bottom pair). On the left are T1 weighted images with gadolinium contrast. On the right are matched T2 weighted images. The images show the changes associated with his previous surgery (the pedicle screw instrumentation has been removed). The full length of relevant spinal cord is difficult to visualize on pre-operative T1 imaging. The 12 month images show no abnormal mass formation or cyst development and no change in the degree of kyphosis.

Fig. 6

Sagittal MR imaging in Patient 2 taken before olfactory ensheathing cell transplantation (top pair) and 12 months afterwards (bottom pair). On the left are T1 weighted images with gadolinium contrast. On the right are matched T2 weighted images. The images show the changes associated with his previous surgery (the pedicle screw instrumentation has been removed). The full length of relevant spinal cord is difficult to visualize on pre-operative T1 imaging. The 12 month images show no abnormal mass formation or cyst development and no change in the degree of kyphosis.

Fig. 7

Sagittal MR imaging of Patient 3 taken before olfactory ensheathing cell transplantation (top pair) and 12 months afterwards (bottom pair). On the left are T1 weighted images with gadolinium contrast. On the right are matched T2 weighted images. Artefact from the titanium vertebral body replacement can be seen anterior to the cord. As in Figs 2 and 3, no masses are evident on the contrast enhanced scans at 12 months and no abnormal fluid cavities have developed. There is no demonstrable change in the volume of the spinal cord.

Fig. 7

Sagittal MR imaging of Patient 3 taken before olfactory ensheathing cell transplantation (top pair) and 12 months afterwards (bottom pair). On the left are T1 weighted images with gadolinium contrast. On the right are matched T2 weighted images. Artefact from the titanium vertebral body replacement can be seen anterior to the cord. As in Figs 2 and 3, no masses are evident on the contrast enhanced scans at 12 months and no abnormal fluid cavities have developed. There is no demonstrable change in the volume of the spinal cord.

Most importantly, no adverse effects of implantation were visible in any of the three patients at 12 months. No change has been visible in the volume of the cystic area at the injury site and syringomyelia has not developed. Pseudomeningocele was not present and no new masses are visible on any of the MRI sequences either at the injection site or at any other point in the neuraxis. The pre-operative sagittal alignment of the spine has been maintained with no new external compression of the spinal cord (the artefact seen in Fig. 4 in the vertebral body space arising from a titanium cage implant which is stable in position).

Discussion

These observations indicate that there were no adverse findings 1 year after transplantation of olfactory ensheathing cells into the injured spinal cord. The neurosurgical procedure did not lead to any negative sequelae either during the operation, or postoperatively. The harvesting and growth of the olfactory ensheathing cells in vitro did not introduce any pathogens into the cultures and their transplantation was not obviously associated with any fever or inflammation. The MRIs at 1 year showed no change from preoperative MRIs indicating no overgrowth of introduced cells and no development of post-traumatic syringomyelia.

The stringent inclusion and exclusion criteria applied to patient selection certainly limited the numbers of participants available. From over 600 enquiries, the numbers were reduced to six participants. Some rejected the trial upon receiving a full briefing and some were rejected for medical reasons. At all stages of selection many were rejected as not fulfilling the required psychosocial criteria including emotional stability, realistic expectations of the trial outcome, stable family and social situation, and lack of drug or alcohol dependence. A strong emphasis was placed on psychosocial selection criteria both for ethical reasons (e.g. not to raise ‘false hopes and expectations’) and to ensure regular participation in the trial for 3 years, an essential criterion for the scientific success of the trial. The trial was initially planned for eight participants but the stringent selection criteria led to slow recruitment of participants, taking 2 years to recruit the final six. Recruitment was stopped at six in order to bring the trial to a close within a reasonable time frame. This difficulty in recruitment reflects on the stringency of selection criteria, the relative numbers of thoracic injuries among the complete paraplegia population, and the relative size of the clinical reach of the local spinal injuries unit. These practical limitations should be considered when designing future clinical trials in order to gain greater numbers of participants. On the other hand, the design of the present study, though small, maximizes statistical power by selecting a homogeneous patient group, a within subjects comparison (i.e. before and after), a non-transplanted control group, a single blind assessment regime, the same assessors throughout, and a rigorous and long-term follow-up.

Syringomyelia was considered an exclusion criterion. In contrast, in a recent trial of foetal spinal cord graft, syringomyelia was an inclusion criterion (Wirth et al., 2001). This relates to the aim of that study to choose an unstable patient population and attempt to stabilize further development of the syrinx (Wirth et al., 2001). It was thought ill-advised to transplant foetal tissue into stable deficits without further information on the risk of causing further impairment (Wirth et al., 2001). This risk was theoretically greater than in the present study because the former included patients with incomplete neurological injury (ASIA B–D) whereas only ASIA A were included here. In other words, there was less function to lose in the present study unless the patients developed a syrinx in the rostral cord. At 1 year none of the patients showed any change on MRI and hence no development of syringomyelia that could be attributable to the transplants.

In general, the overall design of this clinical trial is similar to that endorsed by a recent international workshop on clinical trials in spinal cord injury (Steeves et al., 2004). We considered three inclusion criteria to be paramount: (i) the spinal cord injury should be of traumatic aetiology and be functionally complete; (ii) it should be between T4 and T10 and neurologically stable; and (iii) the participants should be secure from a psycho-social perspective, as well as being able to give fully informed consent.

We chose participants with stable, complete spinal cord injuries at T4–T10. Stable lesions without syringomyelia were chosen so that each patient might act as his own control and thereby increase the power of a study with a necessarily small number of patients. Ideally the pre-operative assessments can be expected to provide a true ‘baseline’ from which to measure changes due to the transplantation. This does not obviate the use of the control participants who are required to control for assessor variability. Assessor variability was also reduced by the use of the same assessors throughout the study, obviating any inter-observer variability. Thoracic injuries were also chosen in order to achieve a more homogeneous patient population because the stability of the thoracic spine provides less variability in the extent of spinal cord dislocation. A consequence of limiting the injuries to these well-defined cases was a considerable reduction in the numbers of cases available from which to choose.

We considered that, in a trial such as this, it would be wise to be in a position to gain information about the efficacy of the procedure if any effect occurred. Thus the selection of complete and neurologically stable spinal cord injury allowed the possibility that any neurological changes that may occur during the study period might reasonably be ascribed to the transplantation procedure and not to idiopathic changes in an incomplete or still recovering injury. Mid-thoracic injuries were chosen to reduce the risk of significant adverse neurological changes should the procedure lead to damage above the site of injury. Clearly any ascending damage to one or two spinal cord segments above an injury would be of lesser consequence in mid-thoracic than in cervical injuries.

The choice of participants with stable psychological status and social situation was considered vital for both ethical and practical reasons. Given the intense public interest and occasional ‘emotionality’ and controversy surrounding spinal cord injury ‘cure’ and regeneration research in addition to the experimental nature of this procedure, it was considered important that serious attempts were made to ensure that the participants were able to comprehend fully the potential benefits (or lack of benefit) and risks associated with the procedure. In other words, that the participants had the personal psychological strength to deal with any outcome or consequence of the study and strong family and social supports to assist them with that process. This was considered especially significant for the control group who, receiving no transplanted cells, had no possibility of improvement but would still be exposed to any public interest that might occur. From a practical perspective, stable psycho-social status was required to maximize attendance at designated follow-up appointments and to avoid any attrition over time that would have reduced the validity of data and placed participants themselves at increased risk if lost to follow-up. This was particularly important given the 3 year follow-up period required for the trial. From the investigator perspective and with only a small number of subjects, it was essential to commence the trial with a strong expectation that all participants would complete it.

We have shown previously the feasibility of the nasal biopsy technique for harvesting olfactory mucosa without affecting the sense of smell (Féron et al., 1998) and the ability to grow olfactory ensheathing cells from these biopsies (Bianco et al., 2004). Transplanted olfactory ensheathing cells were identified in these cultures as positive for GFAP and p75 (>95% and 76–88%, respectively). Because both these proteins are expressed by some non-myelinating Schwann cells (Byers et al., 2004), we checked for Schwann cell contamination using the anti-HNK-1 antibody (Barnett et al., 1993) which labels non-myelinating Schwann cells and myelinating Schwann cells (Martini and Schachner, 1986). All transplanted cells were negative for HNK-1, which is present in cultures of Schwann cells from the trigeminal nerve innervating the olfactory mucosa (Bianco et al., 2004), but because some Schwann cells are HNK-1 negative (Martini and Schachner, 1986) we cannot completely exclude a contribution of Schwann cells in our olfactory ensheathing cell cultures.

Injection of cells or other materials into the spinal cord requires precision and control over the spatial dimensions of the spinal cord and the volume of injectate. Accordingly, the injection system was designed to fulfil these criteria in the operating theatre. The three-dimensional micromanipulator allowed cells to be placed at defined locations and the lever-operated injector device allowed measured volumes of cells to be delivered repeatedly and easily during surgery. The micromanipulator was firmly fixed to the operating table to prevent accidental and unintended movement. The injector device was easily removed from the micromanipulator for refilling using a twist-lock that allowed fast and easy removal and reattachment to the injector device and filling station. The filling station allowed rapid filling of the syringe while preventing the needle from touching the tube containing the cells, thereby maintaining sterility within the surgical field.

The present study demonstrates the feasibility of taking a nasal biopsy and reliably generating enough cells for transplantation within 4 weeks. Of the pathological analyses of the cell cultures, one was positive before transplantation and was discarded. None of the cultures showed signs of contamination when the surplus cells were cultivated after transplantation. These observations and the cell viability assays of the cells at the time of pre-surgical preparation indicate that the cells were viable at the time of transplantation. It is not possible to know if any cells survived in the spinal cords post-transplantation. Methods for labelling cells with ferromagnetic particles (Bulte and Kraitchman, 2004; Dunning et al., 2004), while being useful for MRI evaluation, are not currently approved for clinical use. For this reason we cannot state whether any cells remained within the injection sites immediately after transplantation or at 1 year. If the cells were still present after 1 year, they did not form a discernable mass visible on MRI.

A major advantage of olfactory ensheathing cells as a therapeutic strategy is their ability to be transplanted autologously. In contrast to foetal spinal cord or other strategies, including embryonic stem cells (McDonald et al., 1999), autologous grafting avoids immune rejection and thus avoids immunosuppression that raises further risks and could compromise the effectiveness of the transplanted cells. Accordingly in the current study there was no immunosuppression and recovery from surgery involved no more than the routine treatments, and there was no local inflammation or infection.

These observations suggest that autologous transplantation of olfactory ensheathing cells into the spinal cord is safe, at least at 1 year post-transplantation. Longer-term safety is being monitored for 3 years and will be the subject of a subsequent report.

The authors are grateful to Mr Wayne Monaghan, Supervising Scientist Microbiology, Queensland Health Pathology Service, Princess Alexandra Hospital Campus, for the microbiological sterility testing of the cells prior to transplantation and to Dr Susanne Jeavons, Department of Radiology, Princess Alexandra Hospital, for the radiological assessments. This work was funded by a grant from the Princess Alexandra Hospital Foundation, which had no role in the study design, the collection or analysis of data, the writing of the report or the decision to submit it for publication.

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Author notes

1Eskitis Institute for Cell and Molecular Therapies, Griffith University, Brisbane, 2Queensland Centre for Mental Health Research, The Park Centre for Mental Health, Wacol, 3Department of Otolaryngology, Head and Neck Surgery, 4Department of Orthopaedic Surgery, 5Department of Neurosurgery and 6Spinal Injuries Unit, Queensland Spinal Cord Injuries Service, Princess Alexandra Hospital, Woolloongabba, Queensland, Australia