Research ArticleTissue Engineering

Long-term mechanical function and integration of an implanted tissue-engineered intervertebral disc

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Science Translational Medicine  21 Nov 2018:
Vol. 10, Issue 468, eaau0670
DOI: 10.1126/scitranslmed.aau0670
  • Fig. 1 eDAPS structure and composition after in vivo implantation in the rat tail.

    (A) Representative raw MR images of the first echo of each treatment group (top) and average T2 maps (bottom) of the native disc and eDAPS implants at 10 weeks (10W) and 20 weeks (20W) obtained at 4.7 T. Scale bar, 2 mm. (B and C) Quantification of eDAPS (B) NP and (C) AF T2 values; bars denote significance (P < 0.01). eDAPS biochemical content was further assessed via (D) Alcian blue–stained (proteoglycans) and picrosirius red–stained (collagen) histology sections of 10- and 20-week implants compared with the native rat tail disc space. Scale bars, 500 μm. (E to G) Quantification of glycosaminoglycan (GAG) content in the (E) NP, (F) AF, and (G) PCL EP regions of the eDAPS pre- and post-implantation. (H to J) Quantification of collagen content in the (H) NP (P = 0.01, 20 weeks versus before implantation), (I) AF (P = 0.04, 20W versus before implantation), and (J) PCL EP (P = 0.01, 20W versus before implantation) regions of the eDAPS. Biochemical content is expressed as a percentage of sample wet weight (%WW). Quantitative data are shown as means ± SD (n = 5 to 10 per group for MRI data and n = 3 to 4 per group for biochemistry data). Significant differences between groups were assessed with a Kruskal-Wallis with Dunn’s multiple comparisons test.

  • Fig. 2 Compressive mechanical properties of eDAPS-implanted motion segments in the rat tail.

    (A) Representative stress-strain curves of eDAPS before implantation and after 10 and 20 weeks of implantation. The shaded arrow highlights the maturation of mechanical properties toward native values. (B and C) Quantification of (B) the toe and linear region modulus (P = 0.01, 20-week toe modulus versus preimplantation toe modulus) and (C) transition and maximum strains (*P < 0.01 compared with all groups). Data are shown as means ± SD (n = 4 to 6 per group). Significant differences between groups were assessed with via Kruskal-Wallis with a Dunn’s multiple comparison test. (D) Micro–computed tomography (μCT) scanning before and after the application of physiologic compression in native rat tail motion segments or eDAPS-implanted motion segments from the 20-week group. Color scale is representative of bone density. Scale bar, 500 μm. (E) Axial maps of regional disc height generated from the μCT scans via a custom MATLAB code. The color scale indicates the local disc height. (F) Compressive strain calculated from the average disc height for the native disc and eDAPS under compression. Data are shown as means ± SD (n = 4 per group). Statistical significance between 20-week and native strains was assessed via a two-tailed Mann-Whitney test (P = 0.11).

  • Fig. 3 In vivo integration of eDAPS in the rat tail.

    (A) SHG images of the AF-EP and VB-EP in eDAPS implanted for 10 and 20 weeks. The AF-VB interface of the native rat tail IVD is shown for comparison. Scale bar, 200 μm. (B) Mallory-Heidenhain–stained histology of native rat tail IVD and the PCL EP regions at 10 and 20 weeks. Bone matrix stains purple/pink, unmineralized collagen stains blue, and erythrocytes stain orange (arrows). Scale bar, 200 μm. (C) Representative stress-strain curves from tension to failure tests of eDAPS-implanted motion segments compared to native rat tail motion segments. Two of three motion segments in the 10-week group had quantifiable tensile properties, the remaining sample failed during dissection [represented as “0” data point on graphs (D) and (E)]. (D and E) Quantification of (D) tensile toe and (E) linear region modulus (P = 0.03, 10 weeks versus native). (F and G) Quantification of (F) failure stress (P = 0.01, 10 weeks versus native) and (G) failure strain (P = 0.03, 10 weeks versus native). Quantitative data are shown as means ± SD (n = 3 to 5 per group). Significant differences between groups were assessed using a Kruskal-Wallis with a Dunn’s multiple comparison test.

  • Fig. 4 Translation of eDAPS to a large animal model.

    Photographs of eDAPS sized for the goat cervical disc space fabricated and seeded with bone marrow–derived allogenic MSCs. (A) The C2-C3 disc space was exposed via an anterior approach, and the native disc and portion of the adjacent EPs were removed under distraction. (B) eDAPS (16 mm in diameter and 9 mm in height), prematured for up to 13 weeks, were placed within the prepared disc space, and (C) distraction was released. (D) The motion segment was fixed with a cervical fixation plate. (E) All animals recovered from the procedure without complication and retained full cervical spine function.

  • Fig. 5 Four-week in vivo performance of eDAPS in a goat cervical disc replacement model.

    (A) Alcian blue–stained (proteoglycans) and picrosirius red–stained (collagen) sections of the eDAPS before implantation (after 13 weeks of preculture). (B) Alcian blue– and picrosirius red–stained sagittal histology sections 4 weeks after implantation. Best and worst representative eDAPS are shown. Scale bars, 1 mm. (C) SHG imaging for organized collagen deposition within the PCL EP. Scale bar, 200 μm. (D) DAPI (4′,6-diamidino-2-phenylindole) staining (scale bars, 50 μm) and immunohistochemistry for collagen II, aggrecan, and collagen I in the NP and AF regions of the eDAPS (scale bar, 250 μm).

  • Fig. 6 Eight-week quantitative MRI and mechanical properties of eDAPS in a goat cervical disc replacement model.

    (A and B) Representative T2-weighted MRI images of eDAPS (A) before implantation (scale bar, 2 mm) and (B) 8 weeks after implantation (arrow; scale bar, 5 mm). (C) Quantification of NP T2 relaxation times in eDAPS implants compared to native goat cervical discs [P = 0.04, two-tailed Mann-Whitney test (n = 3 to 13 per group)]. (D) Representative stress-strain curves from compression testing of goat eDAPS before and after implantation compared to native goat cervical motion segments. (E) Quantification of toe and linear moduli of eDAPS-implanted motion segments compared to native goat cervical motion segments and eDAPS before implantation (P = 0.02, preimplantation versus 8-week toe modulus). (F) Quantification of transition and maximum strains in 8-week eDAPS implants compared with native motion segment and eDAPS before implantation (P = 0.04, 8-week versus preimplantation transition strain; P = 0.03, 8-week versus preimplantation maximum strain). Quantitative data are shown as means ± SD. Significant differences in mechanical properties between groups (n = 3 to 4 per group) were assessed via a Kruskal-Wallis with Dunn’s multiple comparison test.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/468/eaau0670/DC1

    Materials and Methods

    Fig. S1. Schematic of eDAPS fabrication and cell seeding for the rat and goat models.

    Fig. S2. EP T2 values of rat eDAPS after implantation.

    Fig. S3. Immunohistochemistry of rat eDAPS after 10 and 20 weeks in vivo.

    Fig. S4. Magnified immunohistochemistry of rat eDAPS after 20 weeks in vivo compared to native.

    Fig. S5. Hematoxylin and eosin staining of rat eDAPS.

    Fig. S6. DAPI staining of rat eDAPS.

    Fig. S7. Histological appearance of goat eDAPS implants from all animals.

    Fig. S8. Hematoxylin and eosin staining of goat eDAPS.

    Fig. S9. Immunohistochemistry of goat eDAPS after 4 weeks in vivo.

    Fig. S10. Sagittal μCT slices of eDAPS after 8 weeks in vivo in the goat cervical spine.

    Movie S1. Goat cervical motion after recovery from eDAPS implantation surgery.

    References (4852)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Schematic of eDAPS fabrication and cell seeding for the rat and goat models.
    • Fig. S2. EP T2 values of rat eDAPS after implantation.
    • Fig. S3. Immunohistochemistry of rat eDAPS after 10 and 20 weeks in vivo.
    • Fig. S4. Magnified immunohistochemistry of rat eDAPS after 20 weeks in vivo compared to native.
    • Fig. S5. Hematoxylin and eosin staining of rat eDAPS.
    • Fig. S6. DAPI staining of rat eDAPS.
    • Fig. S7. Histological appearance of goat eDAPS implants from all animals.
    • Fig. S8. Hematoxylin and eosin staining of goat eDAPS.
    • Fig. S9. Immunohistochemistry of goat eDAPS after 4 weeks in vivo.
    • Fig. S10. Sagittal μCT slices of eDAPS after 8 weeks in vivo in the goat cervical spine.
    • Legend for movie S1
    • References (4852)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Goat cervical motion after recovery from eDAPS implantation surgery.

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