In recent years, cell transplantation has become a focus of attention and reliable outcomes have been achieved in regeneration of the sciatic nerve. The effect of undifferentiated bone marrow stromal cells (BMSCs) on peripheral nerve regeneration was studied using a rat sciatic nerve regeneration model. A 10-mm sciatic nerve defect was bridged using an inside-out vein graft (IOVG) filled with undifferentiated BMSCs (2 × 10 7 cells/ml). In the control group, the vein was filled with phosphate buffer saline alone. The regenerated fibres were studied 4, 8 and 12 weeks after surgery. Assessment of nerve regeneration was based on functional (walking track analysis), histomorphometric and immuohistochemical (Schwann cell detection by S100 expression) criteria. The functional study confirmed significant recovery of regenerated axons in the IOVG/BMSC group ( P < 0.05). Quantitative morphometric analyses of regenerated fibres showed the number and diameter of myelinated fibres in the IOVG/BMSC group were significantly higher than in the control group ( P < 0.05). This demonstrates the potential for using undifferentiated BMSCs in peripheral nerve regeneration without the limitations of donor-site morbidity associated with isolation of Schwann cells. It also reduces costs because the interval between tissue collection and cell injection is reduced and the laboratory procedures are simpler compared to undifferentiated BMSCs.
In spite of the presence of various nerve coaptation materials and techniques, achievement of desired functional peripheral nerve regeneration is still inadequate . Traumatic nerve injury resulting in peripheral nerve gap often requires a graft to bridge the defect. Autologous nerve grafting is the method of choice for bridging peripheral nerve gaps, but it has the disadvantage of sacrificing a functional nerve. Numerous surgical techniques are performed each year for peripheral nerve regeneration . Vein has been used experimentally as a conduit; it has the advantages of no donor morbidity, ease of harvesting and transplanting, availability, affordability and no foreign body reactions .
In the last few years, cell transplantation has become the focus of attention, especially that of Schwann cells, and reliable outcomes have been achieved in the regeneration of the sciatic nerve. It has been shown that transplantation of differentiated bone marrow stromal cells (BMSCs) into silicone tube, vein graft and cut ends of nerves exerts a beneficial effect on peripheral nerve regeneration . To the authors’ knowledge, there is no report of the transplantation of undifferentiated BMSCs into an inside-out vein graft (IOVG) in the literature.
The objective of present study is to elicit functional recovery in peripheral nerve injury over a 10-mm gap defect by undifferentiated fibroblast-like BMSC implantation into IOVG and to evaluate their effectiveness on sciatic nerve regeneration in a rat model. Assessment of nerve regeneration is based on functional (walking track analysis), histomorphometric and immuohistochemical (Schwann cell detection by S-100 expression) criteria, 4, 8 and 12 weeks after surgery.
Materials and methods
Seventy-two male white Wistar rats, weighing approximately 270 g, were separated randomly into four experimental groups ( n = 18): sham-operated group (sham); sciatic nerve amputation group (SNA); control group (IOVG); and undifferentiated BMSC group (IOVG/BMSC). Each group was separated into three subgroups of six animals each. Thirty-six male white Wistar rats weighing 300–350 g were used as vein graft donors. Vein graft harvesting was done from all the rats. Four rats were assigned to isolation and preparation of undifferentiated BMSCs. Two weeks before and during the entire experiment, the animals were housed in individual plastic cages with an ambient temperature of 23 ± 3 °C, stable air humidity, and a natural day/night cycle. The rats had free access to standard rodent laboratory food and tap water.
Isolation and culture of BMSCs
Isolation of adult BMSCs was performed using the donor animals immediately after harvesting of the vein graft according to the method described by C uevas et al. . Rats were killed and their femurs were dissected out. The marrow was extruded with 10 ml of Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 20% foetal bovine serum, and antibiotics (100 U penicillin and 100 mg/ml streptomycin, Gibco, USA). The cells were incubated at 37 °C, humidity 95% and CO 2 5%. After 48 h the non-adherent cells were removed by replacing the medium. The cells were used for the grafting procedure after the cultures had reached confluence. The undifferentiated BMSCs were harvested with 0.25% trypsin and 1 mM EDTA (Gibco, USA), suspended in phosphate buffered saline (PBS) solution and loaded in a 1 ml syringe at a concentration of 2 × 10 7 cells/ml ( Fig. 1 ).
The animals were anaesthetized by intraperitoneal administration of ketamine–xylazine (ketamine 5%, 90 mg/kg and xylazine 2%, 5 mg/kg). The procedures were carried out based on the guidelines of the Ethics Committee of the International Association for the Study of pain . The University Research Council approved all experiments. A 15-mm segment of right external jugular vein was harvested on a tube after the donor animals had been anaesthetized, shaved and prepared aseptically. Grafts were washed in physiological solution and left at room temperature for 30–40 min. A subtle retraction of 1 mm was already expected. Each graft was inverted inside-out to prevent any potential branching of axons through the side branches during regeneration .
Following surgical preparation in the sham group, the left sciatic nerve was exposed through a gluteal muscle incision and after careful hemostasis the muscle was sutured with resorbable 4/0 sutures, and the skin with 3/0 nylon. In the SNA group, the left sciatic nerve was transected proximal to the tibio-peroneal bifurcation where a 7 mm segment was excised, leaving a 10 mm gap due to retraction of nerve ends. Proximal and distal stumps were fixed in the adjacent muscle with 10/0 nylon epineurial suture. No conduit was placed between the stumps. In the IOVG group the left sciatic nerve was exposed through a gluteal muscle incision and transected proximal to the tibio-peroneal bifurcation where a 7-mm segment was excised, leaving a gap about 10 mm due to retraction of nerve ends. Proximal and distal stumps were each inserted 2 mm into the graft and two 10/0 nylon sutures were placed at each end of the cuff to fix the graft in place and leave a 10-mm gap between the stumps. The conduit was filled with 10 μl phosphate buffered saline solution and sterile Vaseline was used to seal the ends of the tubes to avoid leakage. In the IOVG/BMSC group the IOVG was filled with 10 μl aliquots (2 × 10 7 cells/ml).
The animals were anaesthetized (see above) and killed with transcardial perfusion of a fixative containing 2% paraformaldehyde and 1% glutaraldehyde buffer (pH 7.4) 4 ( n = 6), 8 ( n = 6) and 12 weeks ( n = 6) after surgery.
Functional assessment of nerve regeneration
Walking track analysis was performed 4, 8 and 12 weeks after surgery based on B ian et al. . The lengths of the third toe to its heel (PL), the first to the fifth toe (TS), and the second toe to the fourth toe (IT) were measured on the experimental side (E) and the contralateral normal side (N) in each rat. The sciatic function index (SFI) in each animal was calculated using the following formula:
SFI = − 38.3 × EPL − NPL NPL + 109.5 × ETS − NTS NTS + 13.3 × EIT − NIT NIT − 8.8