Total disc replacement using tissue-engineered intervertebral discs in the canine cervical spine

Yu Moriguchi, Jorge Mojica-Santiago, Peter Grunert, Brenton Pennicooke, Connor Berlin, Thamina Khair, Rodrigo Navarro-Ramirez, Rodolfo J Ricart Arbona, Joseph Nguyen, Roger Härtl, Lawrence J Bonassar, Yu Moriguchi, Jorge Mojica-Santiago, Peter Grunert, Brenton Pennicooke, Connor Berlin, Thamina Khair, Rodrigo Navarro-Ramirez, Rodolfo J Ricart Arbona, Joseph Nguyen, Roger Härtl, Lawrence J Bonassar

Abstract

The most common reason that adults in the United States see their physician is lower back or neck pain secondary to degenerative disc disease. To date, approaches to treat degenerative disc disease are confined to purely mechanical devices designed to either eliminate or enable flexibility of the diseased motion segment. Tissue engineered intervertebral discs (TE-IVDs) have been proposed as an alternative approach and have shown promise in replacing native IVD in the rodent tail spine. Here we demonstrate the efficacy of our TE-IVDs in the canine cervical spine. TE-IVD components were constructed using adult canine annulus fibrosis and nucleus pulposus cells seeded into collagen and alginate hydrogels, respectively. Seeded gels were formed into a single disc unit using molds designed from the geometry of the canine spine. Skeletally mature beagles underwent discectomy with whole IVD resection at levels between C3/4 and C6/7, and were then divided into two groups that received only discectomy or discectomy followed by implantation of TE-IVD. Stably implanted TE-IVDs demonstrated significant retention of disc height and physiological hydration compared to discectomy control. Both 4-week and 16-week histological assessments demonstrated chondrocytic cells surrounded by proteoglycan-rich matrices in the NP and by fibrocartilaginous matrices in the AF portions of implanted TE-IVDs. Integration into host tissue was confirmed over 16 weeks without any signs of immune reaction. Despite the significant biomechanical demands of the beagle cervical spine, our stably implanted TE-IVDs maintained their position, structure and hydration as well as disc height over 16 weeks in vivo.

Conflict of interest statement

Competing Interests: LJB and RH hold equity in 3DBio Corp., a private company seeking to develop products related to intervertebral disc regeneration. RH has the following disclosures: Consulting Fees: AOSpine, Brianlab, DePuy-Synthes, Lanx and Supported/Contracted Research: Baxter. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1
A) Schematic picture of disc tissue engineering. NP and AF cells were separately isolated from canine lumbar spine and cultured in vitro. Cultured NP and AF cells were seeded in alginate and collagen gels, respectively, and subsequently both composites were combined into a TE-IVD. B) Photographs of TE-IVD fabrication. Cultured NP cells were injected into a predesigned mold and encircled with two layers of AF cell-laden collagen gels. These AF layers circumferentially contract over cultivation time until they reach a size similar to native IVD as observed in top view and side view. C) Total discectomy and TE-IVD implantation were performed anteriorly under segmental distraction. D) Upon distraction release, stably transplanted TE IVDs remained in place and were secured in the disc space.
Fig 2. X-ray, MRI and histology of…
Fig 2. X-ray, MRI and histology of adjacent motion segment, discectomy, and TE-IVD at 4 and 16 weeks.
Adjacent disc levels showed clear vertebral separation, strong hydration signal in sagittal and axial T2 MRI, highly localized T2 map signal, and abundant staining with Safranin O. Discectomy levels showed no vertebral separation and minimal T2 signal and no soft tissue present between vertebrae. Animals receiving stably transplanted TE-IVD showed clear vertebral separation, with tissue hydration noted in both sagittal and axial T2-weighed MRI (yellow arrows). At both 4 and 16 weeks after transplantation, proteoglycan-rich tissue was observed to be well integrated into the surrounding vertebrae.
Fig 3. Quantitative analysis of disc height…
Fig 3. Quantitative analysis of disc height index and MRI.
A) Stably implanted TE-IVD had significant retention of disc height compared to the discectomy control at 4 and 16 weeks (asterisks, p

Fig 4. Safranin O staining showed an…

Fig 4. Safranin O staining showed an absence of tissue in the intervertebral space of…

Fig 4. Safranin O staining showed an absence of tissue in the intervertebral space of samples in the discectomy group.
Healthy tissue in the adjacent motion segment showed strong proteoglycan staining the NP, with numerous clusters of round chondrocytic cells (open arrows) and less staining in the AF, with more elongated fibrochondrocytes arranged in distinct fibrous lamellae (open arrow heads). At 4 weeks, TE-IVD samples had strong proteoglycan staining in both NP and AF, with rounded cells apparent in the NP (filled arrows) and more elongated cells arranged primitive lamellae in the AF. By 16 weeks, staining was more evident in the NP than AF, with some clustering of rounded chondrocytic cells (filled arrows). AF cells were clearly elongated (closed arrow heads) and aligned. All scale bars are 100 μm.

Fig 5. Picrosirius red staining showed an…

Fig 5. Picrosirius red staining showed an absence of tissue in the intervertebral space of…

Fig 5. Picrosirius red staining showed an absence of tissue in the intervertebral space of samples in the discectomy group.
Healthy tissue in the adjacent motion segment showed little staining for collagen in the NP with numerous clusters of round chondrocytic cells (open arrows) and strong staining in the AF, with more elongated fibrochondrocytes arranged in distinct fibrous lamellae (open arrow heads). At 4 weeks, TE-IVD samples had light collagen staining in the NP and stronger staining in the AF, with rounded cells apparent in the NP (filled arrows) and more elongated cells arranged primitive lamellae in the AF. By 16 weeks, staining was present in both NP than AF, with some clustering of rounded chondrocytic cells (filled arrows) in the NP and elongated cells in the AF (closed arrow heads). All scale bars are 100 μm.

Fig 6. Picrosirius red-stained histology under polarized…

Fig 6. Picrosirius red-stained histology under polarized light showed birefringent features associated with collagen fibers.

Fig 6. Picrosirius red-stained histology under polarized light showed birefringent features associated with collagen fibers.
Discectomy samples show collagen organization in vertebrae, with no tissue in the intervertebral space. Adjacent motion segments show the absence of collagen fibers in the NP and large collagen fibers (~50–100 μm) inserting from the AF to the vertebral body (VB). At 16 weeks, TE-IVD samples show the presence of some small, unorganized collagen fibers in the NP, with larger (~20–50 μm) fibers that insert into the vertebral body (VB).
Fig 4. Safranin O staining showed an…
Fig 4. Safranin O staining showed an absence of tissue in the intervertebral space of samples in the discectomy group.
Healthy tissue in the adjacent motion segment showed strong proteoglycan staining the NP, with numerous clusters of round chondrocytic cells (open arrows) and less staining in the AF, with more elongated fibrochondrocytes arranged in distinct fibrous lamellae (open arrow heads). At 4 weeks, TE-IVD samples had strong proteoglycan staining in both NP and AF, with rounded cells apparent in the NP (filled arrows) and more elongated cells arranged primitive lamellae in the AF. By 16 weeks, staining was more evident in the NP than AF, with some clustering of rounded chondrocytic cells (filled arrows). AF cells were clearly elongated (closed arrow heads) and aligned. All scale bars are 100 μm.
Fig 5. Picrosirius red staining showed an…
Fig 5. Picrosirius red staining showed an absence of tissue in the intervertebral space of samples in the discectomy group.
Healthy tissue in the adjacent motion segment showed little staining for collagen in the NP with numerous clusters of round chondrocytic cells (open arrows) and strong staining in the AF, with more elongated fibrochondrocytes arranged in distinct fibrous lamellae (open arrow heads). At 4 weeks, TE-IVD samples had light collagen staining in the NP and stronger staining in the AF, with rounded cells apparent in the NP (filled arrows) and more elongated cells arranged primitive lamellae in the AF. By 16 weeks, staining was present in both NP than AF, with some clustering of rounded chondrocytic cells (filled arrows) in the NP and elongated cells in the AF (closed arrow heads). All scale bars are 100 μm.
Fig 6. Picrosirius red-stained histology under polarized…
Fig 6. Picrosirius red-stained histology under polarized light showed birefringent features associated with collagen fibers.
Discectomy samples show collagen organization in vertebrae, with no tissue in the intervertebral space. Adjacent motion segments show the absence of collagen fibers in the NP and large collagen fibers (~50–100 μm) inserting from the AF to the vertebral body (VB). At 16 weeks, TE-IVD samples show the presence of some small, unorganized collagen fibers in the NP, with larger (~20–50 μm) fibers that insert into the vertebral body (VB).

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