In Vivo Total Disc Replacement using Tissue-Engineered Intervertebral Discs in a Canine Model

Abstract

Introduction Disc degeneration in the cervical spine is a prevalent clinical predicament often requiring surgery. Anterior cervical decompression and fusion (ACDF), the most commonly performed procedure, poses risks of pseudoarthrosis, and adjacent segment disease (ASD).1,2 An emerging alternative treatment option is prosthetic total disc replacement (TDR),3 which preserves segmental mobility. Our group previously developed a biological TDR device using composite AF/NP disc-like construct with viable cells and mechanical properties analogous to the native discs in a rat tail model.4 In this study, we evaluated the in vivo efficacy of this tissue-engineered intervertebral disc (TE–IVD) in a beagle cervical model assessing radiological and histological parameters. Material and Methods TE-IVD construction: Canine-sized TE–IVDs were constructed as previously described.4 Cervical IVDs from skeletally mature beagles were separated into AF and NP tissues; component cells were isolated and cultured in vitro. Cultured NP cells were seeded with alginate, injected into a predesigned mold, and encircled with two layers of an AF cell laden collagen gel. The combined construct was kept in media for 2 weeks as the surrounding annulus fibrous aligned and contracted until required TE–IVD diameter was attained. Experimental Protocol: Overall, eight skeletally mature beagles were divided into the following two groups: the control group ( n = 2) underwent discectomy with fully resected IVDs, and the experimental group ( n = 6) underwent TE–IVD implantation postdiscectomy. Adjacent proximal segments served as internal healthy controls. Postoperative X-ray and MRI were taken at 2 and 4 weeks; disc height indices5 and NP hydration using a pre-established algorithm6 were analyzed. Beagles were humanely killed at 4 weeks for histological assessment. Results TE–IVDs were successfully implanted postdiscectomy ( Fig. 1A , B). At 2 weeks, MRIs of TE–IVDs revealed T2 high intensity with acute outer inflammation because of the surgical invasion, which faded by 4 weeks. At 4 weeks, TE–IVDs maintained position in the disc space with relatively increased T2 intensity, whereas discectomized segments manifested as black discs ( Fig. 1C , D). These findings suggest that the implanted TE–IVDs engraft in the disc space despite significant biomechanical demands of the beagle cervical environment. In fact, disc height indices of the TE–IVDs and discectomized discs were 71 and 49%, respectively, of that of healthy control discs. Likewise, MRIs revealed that NP hydration of the implanted TE–IVDs was over 70% of that of healthy discs. Histological assessments further demonstrated chondrocyte-like cell viability in the TE–IVD, abundant proteoglycan content in the extracellular matrices, and substantial integration into host tissues without signs of immune reactions. [Figure: see text] Conclusion Despite the severe local milieu of the beagle cervical spine owing to mechanical loading, our in vivo TE–IVDs when appropriately implanted, maintained their position and structure at 4 weeks. The TE–IVDs displayed dynamic adaptation to the host environment, with extracellular matrix production and cell proliferation. They further maintained disc height as well as NP hydration at 4 weeks with up to 70% viability as the normal healthy discs. 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

Promise, progress, and problems in whole disc tissue engineering

“…These findings are similar to the results of implantation of engineered discs of the same design within the rat lumbar spine . In the canine cervical spine, proteoglycan content within the engineered disc appeared to decrease with increasing duration of implantation, and MRI T2 relaxation times in the NP were not significantly different from discectomy after 16 weeks …”

“…Engineered discs of the same design have recently been translated from the rat tail model into a canine cervical disc replacement model, the first study to report the implantation of a tissue‐engineered disc in a large animal model. In contrast to the rat tail implantation studies, where the engineered discs remained in place without the need for fixation, in this larger canine model, displacement of the construct occurred in half of the animals investigated . In successfully retained constructs, however, disc height remained significantly higher than discectomy controls after 16 weeks, and evidence of construct integration with the native tissue was observed via histological analysis.…”

“…However, divergent approaches exist regarding the most appropriate manner in which to culminate in cell‐seeded engineered discs that are mature and mechanically functional in the in vivo environment. Two main strategies have been pursued—maturation of the construct in vitro via pre‐culture prior to in vivo implantation, and implantation of an immature construct without preculture that subsequently undergoes maturation in the in vivo space …”

“…Although it is currently unclear what maturation state an engineered disc should reach in order to achieve native tissue equivalence in vivo within the spine, it is likely that some degree of functional maturation will be necessary. Therefore, several groups have utilized various in vitro culture strategies to stimulate disc‐like matrix deposition and enhance the mechanical function of engineered disc constructs prior to in vivo implantation …”

“…The use of allogeneic disc cells may provide a solution to this issue. Several studies in large animal models have demonstrated minimal immune reaction of allogeneic cells delivered to the disc space within either tissue‐engineered discs or hydrogel carriers, and a clinical trial of allogeneic MSCs for meniscal regeneration also demonstrated no significant immune response to allogeneic cell delivery …”