Tissue-engineered Total Disc Replacement: In vivo Outcomes of a Canine Cervical Disc Study
Authors: Moriguchi Yu , Santiago Jorge Mojica , Navarro-Ramirez Rodrigo , Grunert Peter , Pennicooke Brenton , Berlin Connor , Hudson Katherine , Bonassar Lawrence , Härtl Roger 
Published: April 2016

Introduction Despite being effective, the most commonly performed treatments for degenerative disc disease (DDD), fusion and prosthetic total disc replacement, still pose risks of pseudoarthrosis, implant dislodgement, and adjacent segment disease.1–3 Tissue engineered intervertebral discs (TE-IVD) are an alternative treatment option for DDD, and have been previously developed by our group as a biological TDR device.4 Presently, we evaluate the surgical conditions that promote implant stability and in-vivo efficacy of our TE-IVDs in a translational beagle cervical spine model. Material and Methods TE-IVD Construction: TE-IVD components were constructed in vitro using either annulus fibrosus (AF) or nucleus pulposus (NP) cultivated canine disc cells; the collagen gel based composite AF enclosed an alginate gel based composite NP, as previously described.4 Experimental and Surgical Protocol: 14 skeletally mature beagles underwent discectomy with whole IVD resection at a level between C3/4 and C6/7, and were divided into two groups: a solely discectomized control ( n = 2) and a TE-IVD implanted group ( n = 12). Discectomy and TE-IVD implantation were performed under segmental distraction. Implant stability was evaluated upon distraction release at the end of the procedure. Imaging: Postoperative imaging was performed with conventional X-rays and high-resolution 3-Tesla MRI under full anesthesia. Disc height indices were measured on X-rays using a pre-established method. 5 All MRIs were analyzed both qualitatively and quantitatively in accordance to T2-weighted images. Utilizing a novel algorithm developed by our group, we filtered out all MRI voxels unrepresentative of NP tissue using their T2-relaxation time (T2-RT), sequestering the extent of NP hydration based on the mean T2-RT within the NP voxel.6 Histological assessment: Animals were sacrificed either at 4 or 16 weeks. Histological staining was obtained using Safranin-O for proteoglycans. Statistics: A Chi-Squared test was performed to determine the correlation between implant stability and surgical level or posterior longitudinal ligament (PLL) resection. For the analyses of continuous outcomes in disc height index, NP size, and NP hydration, we employed linear regression models with a generalized estimating equation and robust standard errors to estimate differences in mean changes from baseline controls (discectomy) across displaced and stable implantation groups. Results TE-IVDs that demonstrated displacement of over 25% TE-IVD volume upon distraction release were defined as “displaced” implants and the remaining were termed as “stable” implants. There was a correlation between implant stability and surgical level but not between implant stability and PLL resection, with implants at C3/4 having the greatest stability ( p < 0.05). Quantitative X-ray and MRI assessments showed that only the stable implants had significant retention of disc height and NP size as well as NP physiological hydration compared with discectomy controls. Both 4- and 16-week histology demonstrated that implanted TE-IVDs yielded AF-like and NP-like tissues in the treated segment. Integration into host tissue was confirmed over 16 weeks without any signs of immune reaction. Conclusion Despite significant biomechanical demands of the beagle cervical milieu, our in vivo TE-IVDs, when implanted successfully, maintained their position, structure and hydration in addition to disc height over 16 weeks. References Nesterenko SO, Riley LH III, Skolasky RL. Anterior cervical discectomy and fusion versus cervical disc arthroplasty: current state and trends in treatment for cervical disc pathology. Spine 2012;37(17):1470–1474 Sugawara T, Itoh Y, Hirano Y, Higashiyama N, Mizoi K. Long term outcome and adjacent disc degeneration after anterior cervical discectomy and fusion with titanium cylindrical cages. Acta Neurochir (Wien) 2009;151(4):303–309, discussion 309 Kelly MP, Mok JM, Frisch RF, Tay BK. Adjacent segment motion after anterior cervical discectomy and fusion versus Prodisc-c cervical total disk arthroplasty: analysis from a randomized, controlled trial. Spine 2011;36(15):1171–1179 Bowles RD, Gebhard HH, Härtl R, Bonassar LJ. Tissue-engineered intervertebral discs produce new matrix, maintain disc height, and restore biomechanical function to the rodent spine. Proc Natl Acad Sci U S A 2011;108(32):13106–13111 Kim JS, Kroin JS, Li X, et al. The rat intervertebral disk degeneration pain model: relationships between biological and structural alterations and pain. Arthritis Res Ther 2011;13(5):R165 Grunert P, Hudson KD, Macielak MR, et al. Assessment of intervertebral disc degeneration based on quantitative magnetic resonance imaging analysis: an in vivo study. Spine 2014;39(6):E369–E378