| Background:Intervertebral disc degenerative low back pain has become one of the most important occupation diseases which can affect s of normal human life. Many countries reported that the fare for the treatment and prevention of this kind of disease has caused a huge economic burden to them in recent decades. So the study on the intervertebral disc degeneration is meaningful.Intervertebral disc degeneration has many presentations, such as the lost nucleus pulposus water content, annulus fibrosus rupture, endplate damage. The recent research consider the loss of nutrition is the final common pathway of disc degeneration, which is mainly diffused from the endplate. But at present the study on the genetic, cell therapy treatment are mainly focus on the degeneration of nucleus pulposus(NP) and are mainly applicable to the patients with early, metaphase disc degeneration. In the recent five decades, intervertebral disc allograft transplantation technique has been studied in animal and human examination as a novel treatment therapy and has achieved impressive outcomes. However, the changes of nutrition function after transplantation of intervertebral disc is still not clear.Dynamic Contrast-enhanced Magnetic Resonance Imaging can obtain perfusion and blood supply of organ along with enhance contrast agents, which has been widely used in the identify of benign and malignant tumors. The research in animal and human also prove its effectiveness and feasibility in the observation of diffusion function of intervertebral disc.Objective:1. To investigate and describe the surgical technique for allograft intervertebral disc transplantation without internal fixation in a dog model.2. Observe the diffusion function of allograft disc after transplantation by DCE-MRI technique and pathological observation to investigate the changes of biological function.Methods:1. Twenty-five adult dogs between 10-15 months old were used in this study, weighing between 10-14 kg. No limitation of the gender. X-ray and MRI were taken to evaluate the condition of the intervertebral disc, excluding the sample with obvious deformity and degeneration using Pfirrmann grading system.2. A total of 5 dogs were sacrificed by overdosed Pentobarbital sodium as donors and the L1-L6 intervertebral discs were harvested en bloc and put into the Pre-configured freezing medium. Cryopreservation was taken by the step of 4℃ (12h)、-20℃(2h)、-40℃(2h)、-80℃(2h) and preserved in liquid nitrogen finally. The remaining twenty dogs were used to investigate allograft intervertebral disc transplantation in L5-L6 segment.3. Left lateral and anteroposterior (AP) X-ray images was performed pre-operative and 1,4,6,8,12 weeks after surgery to investigate the position and the healing of the allograft disc. The height of allograft disc was record and represented as x±s. Compare the data post-operative with the pre-operative respectively and analyze it by means of ANOVA. There is significant difference with P< 0.05.4. Examinations of DCE-MRI for the surgical group were performed 4,8,12,24 weeks after allograft disc transplantation. T1-weighted images were obtained using a 3.0 Tesla MR Imager (SIGNA, General Electric, Milwaukee, WI) pre-contrast and post-contrast (after IV injection of gadodiamide in a single dose of 0.3mmol per/kg) at 2,5,10,30,60,90,120,150,180,210 and 240 min. Five regions of interest were chosen from the upper and lower VB, EPZ and the NP. Record the signal of every ROI at each time-in-point, then calculated the enhancement rate and plotted the curve of time-signal intensity. Analyze the characteristic of the curve.5. Haft dogs were sacrificed 12 weeks post-operation and the remains were sacrificed 24 weeks post-operation. The allograft discs were en bloc with 5mm of bone of VB and fixed in the Paraformaldehyde for 2 weeks, decalcified in Perenyi’s solution for 4 weeks, and embedded in paraffin. H&E staining and Masson staining were performed to observed the structure of nucleus pulpopus, endplate zone and annulus fibrosus(AF).Results:1. Choosing the suitable size of allograft and preserving the anterior and posterior longitudinal ligament, contralateral annular fibrosus and appropriate portion of the anterior annulus fibrosus were essential to successfully transplantation without internal fixation. A total of 2 cases of dislocation and 1 case of infection were found in this study, the remaining 17 cases were successful.2. X-ray exam shows the healing of the osteotomies between allograft and VB 3 month post-operation. No subluxation or dislocation was found. The mean height of allograft disc pre-operation was 3.0±0.7mm, and the height 1,4,6,8,12 weeks post-operation were 2.9±0.3mm,2.7±0.6mm,2.7±0.4mm,2.6±0.8mm,2.6±0.7mm respectively, each of them were lower than pre-operation. But no significant difference was found between the height of allograft disc either between pre-operation and any follow-up(P>0.05), or within the follow-up.3. Through gross specimen observation of intervertebral disc allograft we found that healing of the osteotomies has occurred in 12 week after surgery. The healing zone could still be recognized and no immunoreaction was found. The allograft disc of 12-week group and 24-week group both have some degree of degeneration comparing with the control groups such as decrease of the height, partial denaturation of the nucleus pulposus, no boundaries between the annulus fibrosus and nucleus pulposus, breakage in the annulus fibrosus, but there was no significant difference between the two surgical group.4. Through histological observation of the 12-week group we found that the nucleus pulposus tissue structure was loose and in disorder; some breakage at inner part of the annulus fibrosus, but general structure remained intact; thickening of the cartilage endplate. In the 24-week group we found the annulus fibrosus structure was in disorder; the NP tissue partly fibrosis and no obvious degeneration was found in the endplate zone.5. In the curves of time-signal intensity of 4,8,12,24 weeks post-operation, the signal of EPZ, NP and VB both showed two peak value where there was significant difference between them. In the curves of time-signal intensity of 4-week group, the signal of EPZ and NP reached the peak value at 90 min and 120 min respectively then became fluctuant. No second peak value was found and the enhanced speed and the maximum enhanced rate were both lower than the control group. In the curves of time-signal intensity of 8-week group, the signal of EPZ and NP both reached the peak value at 120 min then became fluctuant. No second peak value was found and the enhanced speed and the maximum enhanced rate were both lower than the control group. In the curves of time-signal intensity of 12-week group, the signal of EPZ and NP both reached the peak value at 120 min then reduced gradually. No second peak value was found and the enhanced speed and the maximum enhanced rate Improved rather than the two groups before, but were still lower than the control group. In the curves of time-signal intensity of 24-week group, the signal of EPZ and NP decreased gradually while reached the maximum in 30min and 90min respectively then increased gradually from 150min and reached the second peak value and there was significant difference between the two peaks.Conclusion:1. Locating the L6 transverse processes by palpation pre-operation could ensure the accuracy of operation section. Using appropriate surgical technique may help to the success of the allograft disc transplantation. Choosing appropriate size of the allograft disc was helpful to the success of operation and reduction of the risk of subluxation or dislocation. This study provides a practicable procedure for allograft disc transplantation.2. The diffusion function of allograft disc decreased at 4 and 8 weeks after surgery. The main reason may be the incomplete healing of the osteotomies. No second peak value was found in 240 min because of the incomplete vasoganglion along with the decrease of disc metabolism rate which lead to the delay of diffusion of contrast agent.3. The diffusion function of allograft disc improved at 12 weeks after surgery, while was still lower than the normal disc. It may due to the reconstruction of the vasoganglion between allograft disc and VB which accelerate the diffusion of the contrast agent. However, the nutrition supply of the allograft affected by the surgery may lead to the decrease of the NP cell activity and extracellular matrix. The metabolism function decreased so no second peak value was found.4. The diffusion function of allograft disc almost recover at 24 weeks after surgery. The second peak value appeared at 210 min even the maximum enhanced rate was still lower than control groups from which we can tell that the transplantation surgery didn’t bring any damage to the endplate. Although the transplantation operation has certain influence on disc diffusion function and the NP cell viability in early stage, they will almost return to normal if there is no damage in the endplate.In conclusion, allograft disc transplantation can rebuild the stability, mobility and biological activity as a treatment therapy of degenerative disc disease(DDD). Through the observation of the dynamic contrast-enhanced magnetic resonance imaging post-operation, we found that the diffusion function of the allograft disc was affected in the early stage which can recover because of the rebuild of the vasoganglion, endplate function. This study not only investigated the relation between the endplate function after intervertebral disc allograft and disc diffusion ability, but also provided a reliable basis for the clinical use of this surgical technique in the future. |
PDF Full Text Request