In vitro evaluation of 3D printed polycaprolactone scaffolds with angle-ply architecture for annulus fibrosus tissue engineering

Abstract

Tissue engineering of the annulus fibrosus (AF) is currently being investigated as a treatment for intervertebral disc degeneration, a condition frequently associated with low back pain. The objective of this work was to use 3D printing to generate a novel scaffold for AF repair that mimics the structural and biomechanical properties of the native tissue. Multi-layer scaffolds were fabricated by depositing polycaprolactone struts in opposing angular orientations, replicating the angle-ply arrangement of the native AF tissue. Scaffolds were printed with varied strut diameter and spacing. The constructs were characterized morphologically and by static and dynamic mechanical analyses. Scaffold surfaces were etched with unidirectional grooves and the influence on bovine AF cell metabolic activity, alignment, morphology and protein expression was studied in vitro . Overall, the axial compressive and circumferential tensile properties of the scaffolds were found to be in a similar range to the native AF tissue. Confocal microscopy images indicated that cells were able to attach and spread on the smooth polycaprolactone scaffolds, but the surface texture induced cellular alignment and proliferation. Furthermore, immunofluorescence analysis demonstrated the aligned deposition of collagen type I, aggrecan and the AF-specific protein marker tenomodulin on the etched scaffolds. Overall, results demonstrated the potential for using the scaffolds as a template for AF regeneration.

Oriented inner fabrication of bi-layer biomimetic tendon sheath for anti-adhesion and tendon healing

“…Without the encumbrances such as complexity of the production process, uncertainty of mechanism involving drug delivery, and disturbance of tendon regeneration, the inherent plain structure is one of Recently, oriented materials and their applications in orthopedic tissue engineering have been studied intensively, including peritoneal adhesion, 16 cartilage degeneration, 15 peripheral nerve defect, 13 annulus fibrosus implant, 12 and rotator cuff tendon defect. 14 However, there is still no application in the field of biomaterials for tendon adhesion.…”

An optimized step‐by‐step protocol for isolation of nucleus pulposus, annulus fibrosus, and end plate cells from the mouse intervertebral discs and subsequent preparation of high‐quality intact total RNA

“…As Shh and Bra are expressed only by the NP cells and not by AF cells, 32,33,41 these markers were used to identify NP cell contamination in the AF cell preparations. Tnmd is a specific molecular marker of the AF cells and is not expressed by NP cells in the intervertebral disc 34 . Hence, expression analysis of Tnmd was used to validate the absence of AF cells in the NP cell preparations.…”

“…The following protocol may be used to microdissect and isolate each component of the disc from E16.5 to all ages during the postnatal stage in mice. The purity of the NP and AF samples was validated by analyzing the expression of specific molecular markers using Brachyury ( Bra , Tbxt , or T , 32 ) and sonic hedgehog ( Shh , 33 ) for NP cells, and tenomodulin ( Tnmd , 34 ) for AF cells. While this methods paper describes cell isolation from the lumbar disc, as degeneration and chronic low back pain is highly prevalent at this level of spine in mice, 26,35 we currently use the same methodology for microdissection and isolation of equally pure populations of NP and AF cells from the cervical, thoracic, and caudal discs of postnatal mice of all ages.…”

Intervertebral Disk Degeneration: The Microenvironment and Tissue Engineering Strategies

“…Three-dimensional printing precisely fabricates the scaffold structure. To generate a scaffold with angle-ply architecture similar to natural AF, Christiani et al (2019) printed laminar constructs comprised of polycaprolactone (PCL) struts oriented at alternating angles of ±30°. The mechanical characterization results showed that the mechanical properties of the scaffolds were similar to native AF tissue.…”