Introduction: Additive Manufacturing (AM) opens up fascinating possibilities for the generation of implants and tissue engineering (TE) constructs. 3D plotting, i. e. extrusion of pasty biomaterials at room or physiological temperature, allows utilization of a great variety of materials and incorporation of sensitive substances like drugs, growth factors or even living cells. Multichannel-3D plotting enables processing of more than one material within one construct and therefore manufacturing of complex specimens, e.g. for application at tissue interfaces and/or generation of improved tissue equivalents which closer mimic natural tissues than conventional TE strategies. We wanted to investigate whether we can load calcium phosphate cement (CPC) pastes with growth factors and combine them with biopolymer hydrogels for AM of complex scaffolds. Further questions were whether live cells can be included within the hydrogels and both materials combined in joint strands with core/shell morphology.
Materials and Methods: For 3D plotting BioScaffolder 2.1 from GeSiM (Grosserkmannsdorf, Germany) was used. As CPC paste an hydroxyapatite (HA) forming α-tricalcium phosphate-based precursor mixture, suspended in a biocompatible oily phase[1], was utilized. Blends of alginate (3%) and methylcellulose (9%)[2] were used as hydrogel material. Before scaffold preparation, all materials were sterilized. After plotting of the scaffolds in air they were stabilized by immersion in 100 mM CaCl2 solution. As growth factor, VEGF (vascular endothelial GF) was used and human mesenchymal stroma cells (hMSC) for embedding experiments. For preparation of core/shell strands self-made double-nozzle extruders were applied, mounted onto the 3D plotter. The scaffolds were fully characterized including morphology, mechanical properties, VEGF release (ELISA and bioassay), cell survival (live/dead and MTT staining) and osteogenic differentiation. One type of scaffold was tested in a pilot animal study in a 5 mm femur defect in rat, stabilized with a metal plate.
Results and Discussion: Combination of CPC and biopolymer hydrogel strands within one scaffold was possible.
Immersion of the scaffolds after plotting in calcium ion containing media led to CPC setting and simultaneously to alginate crosslinking. Methylcellulose which improved viscosity and therefore plottability of the hydrogel paste slowly dissolves during further storage in aqueous media, creating an additional microporosity (besides the macropores = space between the strands). We could demonstrate that VEGF could be successfully incorporated in CPC as well as hydrogel strands; for mixing with CPC binding of the GF to chitosan microparticles was advantageous. GF release was much faster from hydrogel compared to CPC – and retained biological activity of released VEGF was proven with an bioassay using endothelial cells. Human MSC could be mixed into the biopolymer hydrogel blend and survived extrusion, scaffold crosslinking and additional 3 weeks of culture. Osteogenic differentiation could be induced by applying common supplements. In addition, the animal pilot study proved biocompatibility of the new type of scaffold and biological activity of the released VEGF in vivo. A novel type of core/shell strands with a stiff shell (CPC) and soft core (hydrogel) and 3D scaffolds made thereof could be manufactured using double-nozzle extruders. Such material combinations within one extruded strand open up new possibilities for dual drug loading and release.
Conclusion: Multichannel 3D plotting is a versatile AM technology and allows combination of such different biomaterials like a calcium phosphate cement and a biopolymer hydrogel. Due to the mild processing conditions sensitive components like GF and living cells can be incorporated. Of special interest is the combination of different biomaterials in strands with core/shell morphology. This technology can be further developed towards more complex tissue and organ models.
Saxon State Ministry for Higher Education, Research and the Arts (SMWK) for funding
References:
[1] A. Lode, K. Meissner, Y. Luo, F. Sonntag, S. Glorius, B. Nies, C. Vater, F. Despang, Th. Hanke, M. Gelinsky: Fabrication of porous scaffolds by 3D plotting of a pasty calcium phosphate bone cement under mild conditions. J. Tissue Eng. Regen. Med. 2014;8:682-693
[2] K. Schütz, A.-M. Placht, B. Paul, S. Brüggemeier, M. Gelinsky, A. Lode: 3D plotting of a cell-laden alginate/methylcellulose blend: towards biofabrication of tissue engineering constructs with clinically relevant dimensions. J. Tissue Eng. Regen Med. 2015 (in press, DOI 10.1002/term.2058)