Bioprinting of mineralized constructs utilizing multichannel plotting of a self-setting calcium phosphate cement and a cell-laden bioink

Abstract

Due to their characteristic resemblance of the mineral component of bone, calcium phosphates are widely accepted as optimal bone substitute materials. Recent research focused on the development of pasty calcium phosphate cement (CPC) formulations, which can be fabricated into various shapes by low-temperature extrusion-based additive manufacturing, namely 3D plotting. While it could be demonstrated that sensitive substances like growth factors can be integrated in such printed CPC scaffolds without impairment of their biological activity live cells cannot be suspended in CPC as they may not be functional when enclosed in a solid and stiff matrix. In contrast, 3D bioprinting of soft cell-laden hydrogels (bioinks) enables the fabrication of constructs with spatially defined cell distribution, which has the potential to overcome problems of conventional cell seeding techniques-but such objects lack mechanical stability. Herein, we combine 3D plotting of CPC and bioprinting of a cell-laden bioink for the first time. As model bioink, an alginate-methylcellulose blend (alg/mc) was used, previously developed by us. Firstly, a fabrication regime was established, enabling optimal setting of CPC and cell survival inside the bioink. As the cells are exposed to the chemical changes of CPC precursors during setting, we studied the compatibility of the complex system of CPC and cell-laden alg/mc for a combined extrusion process and characterized the cellular behavior of encapsulated human mesenchymal stroma cells within the bioink at the interface and in direct vicinity to the CPC. Furthermore, biphasic scaffolds were mechanically characterized and a model for osteochondral tissue grafts is proposed. The manuscript discusses possible impacts of the CPC setting reaction on cells within the bioink and illustrates the advantages of CPC in bioprinting as alternative to the commonly used thermoplasts for bone tissue engineering.

3D Plotted Biphasic Bone Scaffolds for Growth Factor Delivery: Biological Characterization In Vitro and In Vivo

“…However, a reduced cell proliferation in the presence of CPC can be concluded, too (Figure ; Figure S4, Supporting Information). That can be attributed rather to the reduced calcium and/or increased phosphate ion concentration in the medium than to the presence of residues of the carrier liquid …”

“…In order to have a higher amount of VEGF in the scaffold center, CPC strands were gradually substituted by AlgGG strands as depicted in Figure a (type I scaffold) . As the mechanical stiffness of the hydrogel strands is much weaker than that of hydroxyapatite, compressive properties of the biphasic scaffolds are impaired in comparison to those of monophasic CPC scaffolds . However, by applying osteosynthesis plates, fixed with four screws to the remaining bone, the critical size defect should be stabilized and mechanical load induced by movement of the rat compensated and therefore, mechanical strength of a scaffold lower than that of bone should not necessarily be disadvantageous for healing.…”

“…The integration of a growth factor depot consisting of an enzymatically degradable fibrin‐based hydrogel by plotting the growth factor–laden biomaterial ink between the CPC strands in the scaffold center would be an alternative solution that could be realized without reduction of mechanical strength and osteoconductivity of the scaffold as no CPC strands would be replaced and the hydrogel strands are faster degraded making room for ingrowing bone tissue. Recently, we described the further development of our approach by MP of the CPC in combination with a cell‐laden bioink . This enables the integration of osteogenic and vasculogenic progenitor cells in such biphasic scaffolds that might be a more effective, future‐oriented alternative as described recently …”

From Shape to Function: The Next Step in Bioprinting

“…Cell viability was preserved during cement setting and porous triphasic osteochondral constructs were created, composed of i) a bone‐mimetic CPC‐only region, ii) a calcified‐cartilage mimetic region, where MSC‐laden bioinks and CPC strands are alternated, and iii) a bionk only region, to simulate articular cartilage. Adhesion between the three compartments is ensured by the middle layer, in which the alternated cement and bioink strands permit the anchoring to the bone and cartilage region respectively . Osteochondral constructs have in general been studied using a wide array of materials, and recently even strategies to print with soft hydrogel both the bone and cartilage compartments have been enabled for instance through printing into supporting, non‐Newtonian baths, showing potential to preserve in each region osteoblastic and chondrogenic cell phenotypes, greatly increasing the versatility and array of materials available for this type of application.…”

Orthotopic Bone Regeneration within 3D Printed Bioceramic Scaffolds with Region‐Dependent Porosity Gradients in an Equine Model

“…This is a mild reaction that permits co‐printing of such CaP cements with cell‐laden bioinks to generate composite constructs. [ 12 ] This class of materials has already been successfully exploited to obtain printed CaP cements with osteoinductive properties, either by encapsulation of growth factors in the paste precursor [ 13 ] or by tuning printable CaP nano‐topography. [ 14 ] As new three‐dimensionally (3D) printed CDHA‐based scaffolds with controllable macro‐ and microscale architectures are becoming available, it becomes increasingly important to investigate their relative regenerative potential not only in vitro, but also in reliable animal models.…”