Using synchrotron radiation inline phase-contrast imaging computed tomography to visualize three-dimensional printed hybrid constructs for cartilage tissue engineering.

Synchrotron radiation inline phase-contrast imaging combined with computed tomography (SR-inline-PCI-CT) offers great potential for non-invasive characterization and three-dimensional visualization of fine features in weakly absorbing materials and tissues. For cartilage tissue engineering, the biomaterials and any associated cartilage extracellular matrix (ECM) that is secreted over time are difficult to image using conventional absorption-based imaging techniques. For example, three-dimensional printed polycaprolactone (PCL)/alginate/cell hybrid constructs have low, but different, refractive indices and thicknesses. This paper presents a study on the optimization and utilization of inline-PCI-CT for visualizing the components of three-dimensional printed PCL/alginate/cell hybrid constructs for cartilage tissue engineering. First, histological analysis using Alcian blue staining and immunofluorescent staining assessed the secretion of sulfated glycosaminoglycan (GAGs) and collagen type II (Col2) in the cell-laden hybrid constructs over time. Second, optimization of inline PCI-CT was performed by investigating three sample-to-detector distances (SDD): 0.25, 1 and 3 m. Then, the optimal SDD was utilized to visualize structural changes in the constructs over a 42-day culture period. The results showed that there was progressive secretion of cartilage-specific ECM by ATDC5 cells in the hybrid constructs over time. An SDD of 3 m provided edge-enhancement fringes that enabled simultaneous visualization of all components of hybrid constructs in aqueous solution. Structural changes that might reflect formation of ECM also were evident in SR-inline-PCI-CT images. Summarily, SR-inline-PCI-CT images captured at the optimized SDD enables visualization of the different components in hybrid cartilage constructs over a 42-day culture period.

Traditional Invasive and Synchrotron-Based Noninvasive Assessments of Three-Dimensional-Printed Hybrid Cartilage Constructs In Situ.

Three-dimensional (3D)-printed constructs made of polycaprolactone and chondrocyte-impregnated alginate hydrogel (hybrid cartilage constructs) can mimic the biphasic nature of articular cartilage, thus offering promise for cartilage tissue engineering applications. Notably, the regulatory pathway for medical device development requires validation of such constructs through in vitro bench tests and in vivo preclinical examinations for premarket approval. For this, noninvasive imaging techniques are required for effective evaluation of the progress of these cartilage constructs, especially when implanted in animal models or human subjects. However, characterization of the individual components of the hybrid cartilage constructs and their associated time-dependent structural changes by currently available noninvasive techniques is challenging as these constructs contain a combination of hydrophobic and hydrophilic biomaterials with different refractive indices. In this study, we report the use of a novel synchrotron radiation inline phase contrast imaging computed tomography (SR-inline-PCI-CT) approach for noninvasive (in situ) characterization of 3D-printed hybrid cartilage constructs that has been implanted subcutaneously in mice over a 21-day period. In parallel, traditional invasive assays were used to evaluate the in vivo performance of the implanted hybrid cartilage constructs with respect to their cell viability and secretion of cartilage-specific extracellular matrix over the 21-day period postimplantation in mice. SR-inline-PCI-CT allowed striking visualization of the individual components within the 3D-printed hybrid cartilage constructs, as well as characterization of the time-dependent structural changes after implantation. In addition, the relationship between the implanted constructs and the surrounding tissues was delineated. Furthermore, traditional assays showed that cell viability within the cartilage constructs was at least 70% at all three time points, and secretion of alcian blue- and collagen type 2-positive matrices increased progressively over the 21-day period postimplantation. Overall, these results demonstrate that the 3D-printed hybrid cartilage constructs have good in vivo performance and validate their potential for regeneration of articular cartilage in vivo. In addition, SR-inline-PCI-CT has demonstrated potential for longitudinal and noninvasive monitoring of the functionality of 3D-printed hybrid cartilage constructs in a way that is translatable to other soft tissue engineering applications.

Modulating mechanical behaviour of 3D-printed cartilage-mimetic PCL scaffolds: influence of molecular weight and pore geometry.

Three-dimensional (3D)-printed poly(ε)-caprolactone (PCL)-based scaffolds are increasingly being explored for cartilage tissue engineering (CTE) applications. However, ensuring that the mechanical properties of these PCL-based constructs are comparable to that of articular cartilage that they are meant to regenerate is an area that has been under-explored. This paper presents the effects of PCL's molecular weight (MW) and scaffold's pore geometric configurations; strand size (SZ), strand spacing (SS), and strand orientation (SO), on mechanical properties of 3D-printed PCL scaffolds. The results illustrate that MW has significant effect on compressive moduli and yield strength of 3D-printed PCL scaffolds. Specifically, PCL with MW of 45 K was a more feasible choice for fabrication of visco-elastic, flexible and load-bearing PCL scaffolds. Furthermore, pore geometric configurations; SZ, SS, and SO, all significantly affect on tensile moduli of scaffolds. However, only SZ and SS have statistically significant effects on compressive moduli and porosity of these scaffolds. That said, inverse linear relationship was observed between porosity and mechanical properties of 3D-printed PCL scaffolds in Pearson's correlation test. Altogether, this study illustrates that modulating MW of PCL and pore geometrical configurations of the scaffolds enabled design and fabrication of PCL scaffolds with mechanical and biomimetic properties that better mimic mechanical behaviour of human articular cartilage. Thus, the modulated PCL scaffold proposed in this study is a framework that offers great potentials for CTE applications.