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

Abstract

Tissue engineering holds great promise for the treatment of advanced intervertebral disc degeneration. However, assessment of in vivo integration and mechanical function of tissue-engineered disc replacements over the long term, in large animal models, will be necessary to advance clinical translation. To that end, we developed tissue-engineered, endplate-modified disc-like angle ply structures (eDAPS) sized for the rat caudal and goat cervical spines that recapitulate the hierarchical structure of the native disc. Here, we demonstrate functional maturation and integration of these eDAPS in a rat caudal disc replacement model, with compressive mechanical properties reaching native values after 20 weeks in vivo and evidence of functional integration under physiological loads. To further this therapy toward clinical translation, we implanted eDAPS sized for the human cervical disc space in a goat cervical disc replacement model. Our results demonstrate maintenance of eDAPS composition and structure up to 8 weeks in vivo in the goat cervical disc space and maturation of compressive mechanical properties to match native levels. These results demonstrate the translational feasibility of disc replacement with a tissue-engineered construct for the treatment of advanced disc degeneration.

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 (upper), and average T2 maps (lower) of the native disc and eDAPS implants at 10 and 20 weeks (scale = 2 mm), obtained at 4.7T. 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 (proteoglycans) and picrosirius red (collagen) stained histology sections of 10 week and 20 week implants compared with the native rat tail disc space (scale = 500 μm). (E) Quantification of GAG content in the NP, (F) AF and (G) PCL endplate regions of the eDAPS. (H) Quantification of collagen content in the NP (P = 0.01, 20W vs. pre-implantation), (I) AF (P = 0.04, 20W vs. pre-implantation) and (J) PCL endplate regions of the eDAPS (P = 0.01, 20W vs. pre-implantation). Quantitative data are shown as mean with standard deviation (n = 5-10 per group for MRI data and n = 3-4 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 prior to implantation, and after 10 and 20 weeks of implantation. The shaded arrow highlights the maturation of mechanical properties towards native values. (B) Quantification of the toe and linear region modulus (P = 0.01, 20W toe modulus vs. pre-implantation toe modulus), and (C) transition and maximum strains (* = P < 0.01 compared with all groups). Data are shown as mean with standard deviation (n = 4-6 per group). Significant differences between groups were assessed with via Kruskal-Wallis with a Dunn’s multiple comparison test. (D) 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 = 500 μm. (E) Axial maps of regional disc height generated from the μCT scans via a custom MATLAB code. Color scale indicates local disc height. (F) Compressive strain calculated from the average disc height for the native disc and eDAPS under compression. Data is shown as mean with standard deviation (n = 4 per group). Statistical significance between 20W 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) Second Harmonic Generation (SHG) images of the AF-endplate and vertebral body (VB)-endplate in eDAPS implanted for 10 and 20 weeks. The AF-vertebral body interface of the native rat tail IVD is shown for comparison. Scale = 200 μm. (B) Mallory-Heidenhain stained histology of native rat tail IVD and the PCL endplate regions at 10 and 20 weeks. Bone matrix stains purple/pink, unmineralized collagen stains blue, and erythrocytes stain orange (arrows). Scale = 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 out 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-E). (D) Quantification of tensile toe and (E) linear region modulus (P = 0.03, 10W vs. native) (F) Quantification of failure stress (P = 0.01, 10W vs. native) and (G) failure strain (P = 0.03, 10W vs. native). Quantitative data are shown as mean with standard deviation (n = 3-5 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 endplates were removed under distraction. (B) 16 mm diameter by 9 mm high eDAPS, pre-matured 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 (proteoglycans) and picrosirius red (collagen) stained sections of the eDAPS prior to implantation (after 13 weeks of pre-culture). (B) Alcian blue and picrosirius red stained sagittal histology sections 4 weeks post-implantation. Best and worst representative eDAPS are shown. Scale = 1 mm. (C) SHG imaging for organized collagen deposition within the PCL endplate, Scale = 200 μm. (D) DAPI staining (scale = 50 μm) and immunohistochemistry for collagen II, aggrecan, and collagen I in the NP and AF regions of the eDAPS (scale = 250 μm).

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

(A) Representative T2-weighted MRIs of eDAPS prior to implantation (scale = 2 mm) and (B) 8 weeks post-implantation (arrow, scale = 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 = 3-13 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 pre-implantation (P = 0.02, pre-implantation vs. 8W toe modulus). (F) Quantification of transition and maximum strain in 8 week eDAPS implants compared with native motion segment and eDAPS pre-implantation (P = 0.04, 8W vs. pre-implantation transition strain; P = 0.03, 8W vs. pre-implantation maximum strain). Quantitative data are shown as mean with standard deviation. Significant differences in mechanical properties between groups (n = 3-4 per group) were assessed via a Kruskal-Wallis with Dunn’s multiple comparison test.