Development of a tissue-engineered composite implant for treating traumatic paraplegia in rats

Abstract

This study was designed to assess a new composite implant to induce regeneration of injured spinal cord in paraplegic rats following complete cord transection. Neuronal xenogeneic cells from biopsies of adult nasal olfactory mucosa (NOM) of human origin, or spinal cords of human embryos, were cultured in two consecutive stages: stationary cultures in a viscous semi-solid gel (NVR-N-Gel) and in suspension on positively charged microcarriers (MCs). A tissue-engineered tubular scaffold, containing bundles of parallel nanofibers, was developed. Both the tube and the nanofibers were made of a biodegradable dextran sulphate-gelatin co-precipitate. The suturable scaffold anchored the implant at the site of injury and provided guidance for the regenerating axons. Implants of adult human NOM cells were implanted into eight rats, from which a 4 mm segment of the spinal cord had been completely removed. Another four rats whose spinal cords had also been transected were implanted with a composite implant of cultured human embryonic spinal cord cells. Eight other cord-transected rats served as a control group. Physiological and behavioral analysis, performed 3 months after implantation, revealed partial recovery of function in one or two limbs in three out of eight animals of the NOM implanted group and in all the four rats that were implanted with cultured human embryonic spinal cord cells. Animals of the control group remained completely paralyzed and did not show transmission of stimuli to the brain. The utilization of an innovative composite implant to bridge a gap resulting from the transection and removal of a 4 mm spinal cord segment shows promise, suggesting the feasibility of this approach for partial reconstruction of spinal cord lesions. Such an implant may serve as a vital bridging station in acute and chronic cases of paraplegia.

Fig. 1

A tubular scaffold containing nanofibers. Original magnification 25 times

Fig. 2

Preparation of adult human NOM and human embryonic SC cultures

Fig. 3

Cultured adult human NOM neurons. a–d Sprouting of nerve fibers concomitantly with migration of nerve cells from M-cell aggregates in NVR-N-Gel. a–c—200 times, d—100 times. a–d Phase contrast microscopy. Immunofluorescent staining of NOM neurons with antibodies specific to MAP 2 (e) and olfactory mucosa protein (OMP) (f). Original magnification e—400 times, f—200 times, respectively

Fig. 4

Phase contrast microscopy of mature motor neuron in a and myelinated axons in b (arrows) in long-term cultures of human embryonic spinal cord cells. Original magnification 400 times

Fig. 5

a Complete paralysis of both legs, folded inward, of a control rat that underwent complete transection of the spinal cord and removal of a 4 mm segment. b Paraplegic rat showing restoration of partial gait performance (in the right leg) 3 weeks after implantation of a composite implant containing cultured adult human NOM cells into a 4 mm gap of transected spinal cord

Fig. 6

a Absence of spinal cord conductivity (SSEP) in a paraplegic control rat after complete transection of the spinal cord and removal of 4 mm segment. b Restoration of spinal cord conductivity after complete transection and implantation of composite implant containing NOM

Fig. 7

q-Space displacement maps (x direction) sequential slices of MRI analyses

Fig. 8

Histological sections of implanted spinal cords 10 months (a–c) after adult NOM implantation and 3 months (d) after implantation of human embryonic spinal cord cells. Hematoxylin-eosin (H&E) staining demonstrates dispersed neuronal perikarya (a, arrows). Silver staining demonstrates nerve fibers—either single (b, arrows), or organized in parallel bundles (d, arrows). In addition, note areas of neurokeratin (c, arrows). Original magnification 400 times