Research ArticleTissue Engineering

Combined nucleus pulposus augmentation and annulus fibrosus repair prevents acute intervertebral disc degeneration after discectomy

See allHide authors and affiliations

Science Translational Medicine  11 Mar 2020:
Vol. 12, Issue 534, eaay2380
DOI: 10.1126/scitranslmed.aay2380
  • Fig. 1 Individual and combined NP and AF repair strategies in an in vivo sheep lumbar spine model.

    Schematics and intraoperative images depicting the tissue-engineered approach to IVD repair. All intraoperative images are oriented with the cranial side of the sheep on the left, caudal on the right, ventral on the top, and dorsal on the bottom. Through a pre-psoas lateral approach to the lumbar spine, IVDs were exposed (A), and those receiving the discectomy injury were subjected to a 3 mm by 10 mm box annulotomy followed by removal of ~200 mg of NP tissue (B). IVDs receiving the biomaterial repair strategies were injected with the modified HA into the NP space (C), had a collagen patch injected and cross-linked in the AF defect (D), or had both treatments. X-linking, cross-linking.

  • Fig. 2 Six-week gross morphological and MRI assessment of individual and combined NP and AF repair.

    (A to E) Axial schematic diagrams of the intact, discectomy only, and treated IVDs. (F to J) Representative sagittal gross dissection images showing the AF at the site of discectomy and biomaterial injections (yellow arrows) after 6 weeks in vivo. (K to O) Representative axial T2 MR images of the IVDs obtained at 3 T showing the AF, NP, and treatment site (yellow arrows). (P to T) Representative mid-sagittal T2 MR images showing differences in the NP across treatment groups. High-intensity regions in T2 MR images correlate with greater water content. (U) Pfirrmann grading of IVD degeneration and (V) DHI from the T2 MR images compared between all groups; bars denote significance (P < 0.05). Pfirrmann grades are shown as median ± interquartile range, whereas DHI is means ± SD (n = 8). Significant differences were assessed with a Kruskal-Wallis with Mann-Whitney U tests for Pfirrmann grades and a general linear mixed model with Tukey’s post hoc tests for DHI.

  • Fig. 3 Six-week histological evaluation of individual and combined NP and AF repair to show overall IVD morphology.

    (A to E) Axial schematic diagrams of the intact, discectomy only, and treated IVDs showing the approximate mid-coronal section through the IVD for histological analyses. (F to J) Representative alcian blue–stained histological sections imaged with bright-field microscopy. Proteoglycans are stained blue, and collagen is stained pink. (K to O) Representative second harmonic generation (SHG) multiphoton images showing collagen orientation and alignment in the IVDs with higher-magnification images to show AF lamellae (P to T). White arrows point to AF lamellae.

  • Fig. 4 Six-week histological evaluation of the discectomy site demonstrating herniation in IVDs without collagen AF repair.

    (A to E) Axial schematic diagrams of the intact, discectomy only, and treated IVDs showing the approximate section through the defect location for histology. (F to J) Representative alcian blue–stained histological sections through the defect location imaged with bright-field microscopy, with higher-magnification images (K to O) showing the intact AF (K), vacuolated chondrocyte-like cells in herniated NP (L and M), and fibrous tissue where the collagen patch was injected (N and O). Alcian blue stains proteoglycans blue and collagen pink, black arrows show direction of collagen alignment in the AF, and white arrows point to chondrocyte-like cells in herniated regions.

  • Fig. 5 Six-week functional mechanical evaluation of the individual and combined NP and AF repairs.

    (A) The mechanical testing protocol encompassing cyclic compression/tension, compressive stress relaxation, and cyclic torsion performed to evaluate mechanical function of the intact, discectomy, and treated IVDs. (B to D) Photographs and schematics of the mechanical testing protocol. IVDs were excised from the spine, stripped of bony processes, potted in PMMA, and loaded into custom fixtures on a biaxial mechanical load frame. (E to I) Representative hysteresis curves for each experimental group obtained from the cyclic torsion loading after preconditioning. (J to L) Torsional stiffness and torque range were quantified from each hysteresis curve, normalized to the intact control from the same sheep, and compared between all groups; bars denote significance (P < 0.05). Quantitative data are shown as means ± SD (n = 3 to 4). Significant differences were assessed using a general linear mixed model with Tukey’s post hoc tests.

  • Fig. 6 Six-week axial loading and stress relaxation mechanical evaluation.

    (A to E) Representative hysteresis curves for each experimental group obtained from the cyclic compressive/tensile loading after preconditioning. (F to I) Compressive stiffness, tensile stiffness, and range of motion were quantified from each hysteresis curve and compared between all groups; bars denote significance (P < 0.05). (J to M) Equilibrium modulus, instantaneous modulus, and hydraulic permeability were quantified from the stress relaxation steps using a poroelastic model fit and compared between all groups; bars denote significance (P < 0.05). Bar plots are shown as means ± SD (n = 3 to 4). Significant differences were assessed using a general linear mixed model with Tukey’s post hoc tests.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/534/eaay2380/DC1

    Materials and Methods

    Fig. S1. Safranin O histology of individual and combined NP and AF repair.

    Fig. S2. Mid-coronal alcian blue histology of the most and least damaged IVDs from each experimental group.

    Fig. S3. SHG imaging of the outer AF from mid-coronal histology sections.

    Fig. S4. Alcian blue histology through the defect site of the most and least damaged IVDs from each experimental group.

    Fig. S5. Hematoxylin and eosin staining of the NP, AF, and endplate near the discectomy site.

    Fig. S6. SHG imaging of histology sections through the defect site.

    Data file S1. Raw data.

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Safranin O histology of individual and combined NP and AF repair.
    • Fig. S2. Mid-coronal alcian blue histology of the most and least damaged IVDs from each experimental group.
    • Fig. S3. SHG imaging of the outer AF from mid-coronal histology sections.
    • Fig. S4. Alcian blue histology through the defect site of the most and least damaged IVDs from each experimental group.
    • Fig. S5. Hematoxylin and eosin staining of the NP, AF, and endplate near the discectomy site.
    • Fig. S6. SHG imaging of histology sections through the defect site.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

Stay Connected to Science Translational Medicine

Navigate This Article