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. 2017 Nov 17;7:15765. doi: 10.1038/s41598-017-15887-4

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Culture and Implantation of DAPS with Engineered Endplates (eDAPS). Given the evidence of poor integration seen in the above studies, DAPS were combined with porous polymer endplates to evaluate whether endplates could bridge the DAPS/vertebra interface and improve tissue integration. The following acronyms are used: EP: endplate, NP: nucleus pulposus, AF: annulus fibrosus, VB: vertebral body. (A) Study design: AF and NP regions were separately seeded and cultured, and then combined along with the acellular endplates to form eDAPS. eDAPS were implanted into the tails of athymic rats and evaluated by MRI, histology, and mechanical testing over 5 weeks in vivo. (B) µCT: 3D µCT reconstruction of an acellular eDAPS, with cut-away illustrating the lamellar AF structure and porous endplate. (C) MRI: (left) Population average T2 maps of DAPS after 1, 2.5, and 5 weeks of implantation. (right) Mean T2 relaxation time of eDAPS AF and NP regions. eDAPS maintained a higher T2 relaxation time 5 weeks after implantation. (**p < 0.001 vs. DAPS at that time point). (D) Histological analysis: Alcian blue/picrosirius red-stained sections of DAPS after implantation. The endplate regions gradually filled with tissue over 5 weeks, with positive collagen staining throughout, while the AF and NP regions maintained PG positive staining over the 5-week implantation period. (D, ‘Interfaces’ top) The endplate/vertebra boundary was bridged with new collagenous tissue that had formed after removal of the vertebral endplate. (D, ‘Interfaces’ bottom) The AF/endplate interface was also bridged by collagenous tissue that was contiguous with tissue within the AF interlamellar spaces. (E) Staining for Cell Nuclei: DAPI staining of the AF, NP, and AF/endplate and vertebra/endplate interfaces. The NP region was filled with rounded cell nuclei typical of cells cast in a hydrogel, while the AF region was filled with elongated cell nuclei in the interlamellar spaces. (E, ‘Interface’ left) The vertebra/endplate interface showed evidence of cells in both regions, suggesting that cells from the vertebrae had crossed the interface into the endplate. (E, ‘Interface’ right) Similarly, The AF/endplate interface showed evidence of cells in both regions, where AF cells had migrated into the endplate region. (F) Mechanical function: Mechanical properties of eDAPS were similar to DAPS at lower strains (toe region modulus, transition strains). At higher strains, eDAPS were less stiff than DAPS without endplates (linear region modulus, maximum strain) though still mechanically robust and comparable to native discs (linear region modulus = 4.4 ± 2.6 MPa). (native rat caudal disc values are shown as dotted lines, + p < 0.1 vs. DAPS; *p < 0.05 vs. DAPS). (G) µCT: There was evidence of early ossification in the eDAPS engineered endplate region.