Biological Responses of Ceramic Bone Spacers Produced by Green Processing of Additively Manufactured Thin Meshes

Abstract

Bone spacers are exclusively used for replacing the tissue after trauma and/or diseases. Ceramic materials bring positive opportunities to enhance greater osteointegration and performance of implants, yet processing of porous geometries can be challenging. Additive Manufacturing (AM) opens opportunities to grade porosity levels in a part; however, its productivity may be low due to its batch processing approach. The paper studies the biological responses yielded by hydroxyapatite with β-TCP (tricalcium phosphate) ceramic porous bone spacers manufactured by robocasting 2-layer meshes that are rolled in green and sintered. The implants are assessed in vitro and in vivo for their compatibility. Human bone marrow mesenchymal stem cells attached, proliferated and differentiated on the bone spacers produced. Cells on the spacers presented alkaline phosphatase staining, confirming osteogenic differentiation. They also expressed bone-specific COL1A1, BGAP, BSP, and SPP1 genes. The fold change of these genes ranged between 8 to 16 folds compared to controls. When implanted into the subcutaneous tissue of rabbits, they triggered collagen fibre formation and mild fibroblastic proliferation. In conclusion, rolled AM-meshes bone spacers stimulated bone formation in vitro and were biocompatible in vivo. This technology may give the advantage to custom produce spacers at high production rates if industrially upscaled.

Keywords: additive manufacturing; bioceramics; biological responses; bone tissue engineering; cell proliferation; implants; porous scaffolds.

Figure 1

Ceramic ink column constructed adding 40 circular horizontal layers of extruded material with a Robocasting syringe 3D printer (Fundació Privada Centre CIM, Barcelona, Spain) at Centre CIM: (a) Elevation view; (b) Isometric view.

Figure 2

Flow diagram of the study undertaken.

Figure 3

Exemplary images: (a) Robocasting system 3D printing the first layer of a squared ceramic mesh; (b) Detail of a thin 3D printed ceramic mesh composed of two layers of material.

Figure 4

Thin 3D printed mesh of the study once rolled into a bone spacer cylinder shape: (a) side view—rolled, in green; (b) top view—final part after heat treatments.

Figure 5

Morphological investigation of a sample prepared for implantation: (a) SEM image acquired at low magnification (60×); (b) SEM image acquired at high magnification (5000×).

Figure 6

Cell adhesion and proliferation of the material and the control groups (Origami refers to the samples produced in the process addressed in the present article).

Figure 7

Fluorescent microscopy picture showing the attachment of the cells to the surface of the material.

Figure 8

Osteogenic differentiation in the material and the control groups in terms of Alkaline Phosphate Yellow Liquid substrate system absorbance.

Figure 9

n-fold changes of bone-specific genes in the material and the control groups.

Figure 10

Representative micrographs from the histologic examination of sections: (A) implanted with PMMA, Haematoxylin & Eosin 200×; (B) implanted with PMMA, Masson’s Trichrome 200× (C) implanted with experimental group, Haematoxylin & Eosin 200×; (D) implanted with experimental group, Masson’s Trichrome 200×. CF: Collagen fibres, **: Granulation Tissue.