3D Bioprinting for Bone Regeneration

There has been an uptick in research activities focusing on 3D bioprinting for bone regeneration. In this issue of “From Academia”, we included four recent publications tackling the issue from different angles. The first article focuses on a new 3D printing composite using silicone resing derived larnite/C scaffold to create new regeneration and treatment strategies for bone tumor patients. The second study focuses on a new bio-ink for 3D bioprinting bone, nanoengineered ionic covalent entanglement (NICE) bioink formulation, which not only showed good printability, mechanical properties, biodegradability, but also the ability to induce endochondral differentiation of encapsulated human mesenchymal stem cells (hMSCs) in the absence of an osteoinductive agent on a genetic level. In the third study, researchers developed a new PLA-based composite formulation that could be used to produce bone scaffold and regeneration. In the last article, a new iron-based ink formulation, as well as matching 3D printing, de-binding, and sintering conditions, was developed to create iron scaffolds with a porosity of 67%, pore interconnectivity of 96%, and a strut density of 89% after sintering. The study shows the great potential of extrusion-based 3D printed porous iron to be further developed as a biodegradable bone substituting biomaterial. “From Academia” features recent, relevant, close to commercialization academic publications. Subjects include but not limited to healthcare 3D printing, 3D bioprinting, and related emerging technologies.

Email: Rance Tino (tino.rance@gmail.com) if you want to share relevant academic publications with us.

Silicone resin derived larnite/C scaffolds via 3D printing for potential tumor therapy and bone regeneration

Authored by Shengyang Fu, Haoran Hu, Jiajie Chen, Yufang Zhu, Shichang Zhao. Chemical Engineering Journal. 15 February 2020

Schematic illustration for the fabrication process of the larnite/C scaffolds and their functions for tumor therapy and bone regeneration. Copyright Chemical Engineering Journal
Schematic illustration for the fabrication process of the larnite/C scaffolds and their functions for tumor therapy and bone regeneration. Copyright Chemical Engineering Journal

Abstract: 

Three-dimensional (3D) printing has been used to fabricate bioceramic scaffolds for treating tumor-related defects in recent years, but the fabrication process and the introduction of anti-tumor agents are still challenging.

In this study, porous free carbon-embedding larniteIn this study, porous free carbon-embedding larnite (a calcium silicate mineral with formula: Ca₂SiO₄. ) (larnite/C) scaffolds have been successfully fabricated by 3D printing of the silicone resin loaded with CaCO3 filler and high-temperature treatment under an inert atmosphere. The fabricated larnite/C scaffolds had uniform interconnected macropores (ca. 400 μm) and exhibited excellent photothermal effect, which was able to kill human osteosarcoma cells (MNNG/HOS) and inhibit the tumor growth in nude mice.

Moreover, the larnite/C scaffolds could stimulate the expression of the osteogenesis-related genes (ALP, OCN, and Runx-2) in rat bone mesenchymal stem cells (rBMSCs), and also promoted new bone formation in critical-sized rat calvarial defects. Therefore, the combination of 3D printing with polymer-derived ceramics strategy could fabricate multifunctional bioceramic scaffolds, which would be promising for potential application in treating tumor-related bone defects.

In vivo osteogenesis performance of the larnite/C scaffolds. (a) 3D reconstruction of micro-CT images. (b) BMD and (c) BV/TV for the larnite and larnite/C-3 groups. (d) New bone formation and mineralization determined histomorphometrically by fluorochrome-labeling analysis. Yellow represents tetracycline at week 2, red represents alizarin red at week 4, green represents calcein at week 6 (scale bar: 100 μm). (e) Histological analysis stained by H&E and Masson (scale bar: 500 μm,). (f) Immunohistochemical staining evaluation of OCN and Runx-2 proteins (scale bar: 100 μm). (* indicated significant difference, p < 0.05). Copyright Chemical Engineering Journal
In vivo osteogenesis performance of the larnite/C scaffolds. (a) 3D reconstruction of micro-CT images. (b) BMD and (c) BV/TV for the larnite and larnite/C-3 groups. (d) New bone formation and mineralization determined histomorphometrically by fluorochrome-labeling analysis. Yellow represents tetracycline at week 2, red represents alizarin red at week 4, green represents calcein at week 6 (scale bar: 100 μm). (e) Histological analysis stained by H&E and Masson (scale bar: 500 μm,). (f) Immunohistochemical staining evaluation of OCN and Runx-2 proteins (scale bar: 100 μm). (* indicated significant difference, p < 0.05).Copyright Chemical Engineering Journal

Nanoengineered Osteoinductive Bioink for 3D Bioprinting Bone Tissue

Authored by David Chimene, Logan Miller, Lauren M. Cross, Manish K. Jaiswal, Irtisha Singh, and Akhilesh K. Gaharwar. ACS Applied Materials & Interfaces. 24 February 2020 

NICE bioink design and printability assessment. (A) The combination of gelatin methacrylate (GelMA), kappa-carrageenan (kCA), and nanosilicates (nSi) was used to design nanoengineered ionic-covalent entanglement (NICE) bioink. (B) Different compositions of NICE formulation was investigated . (C) The 3D printability of each NICE bioink formulation was quantified using screw-driven extrusion printer ( at 37 °C) to fabricate a 3 cm tall, 1 cm wide hollow tube. The effect of different component on printability was evaluated. (D) Print success of 3D printed structure was based on the final height of the structure, conformity to expected dimensions, and lack of observable errors. Copyright ACS Applied Materials and Interfaces
NICE bioink design and printability assessment. (A) The combination of gelatin methacrylate (GelMA), kappa-carrageenan (kCA), and nanosilicates (nSi) was used to design nanoengineered ionic-covalent entanglement (NICE) bioink. (B) Different compositions of NICE formulation was investigated . (C) The 3D printability of each NICE bioink formulation was quantified using screw-driven extrusion printer ( at 37 °C) to fabricate a 3 cm tall, 1 cm wide hollow tube. The effect of different component on printability was evaluated. (D) Print success of 3D printed structure was based on the final height of the structure, conformity to expected dimensions, and lack of observable errors. Copyright ACS Applied Materials and Interfaces

Abstract: 

Bioprinting is an emerging additive manufacturing approach to the fabrication of patient-specific, implantable three-dimensional (3D) constructs for regenerative medicine.

However, developing cell-compatible bioinks with high printability, structural stability, biodegradability, and bioactive characteristics is still a primary challenge for translating 3D bioprinting technology to preclinical and clinal models. To overcome this challenge, we developed a nanoengineered ionic covalent entanglement (NICE) bioink formulation for 3D bone bioprinting.

The NICE bio-inks allow precise control over printability, mechanical properties, and degradation characteristics, enabling custom 3D fabrication of mechanically resilient, cellularized structures. We demonstrate cell-induced remodeling of 3D bioprinted scaffolds over 60 days, demonstrating deposition of nascent extracellular matrix proteins. Interestingly, the bioprinted constructs induce endochondral differentiation of encapsulated human mesenchymal stem cells (hMSCs) in the absence of an osteoinductive agent.

Using next-generation transcriptome sequencing (RNA-seq) technology, we establish the role of nanosilicates, a bioactive component of NICE bioink, to stimulate endochondral differentiation at the transcriptome level. Overall, the osteoinductive bioink has the ability to induce the formation of osteo-related mineralized extracellular matrix by encapsulated hMSCs in growth factor-free conditions.

Furthermore, we demonstrate the ability of NICE bioink to fabricate patient-specific, implantable 3D scaffolds for repair of craniomaxillofacial bone defects. We envision the development of this NICE bioink technology toward a realistic clinical process for 3D bioprinting patient-specific bone tissue for regenerative medicine.

Extracellular matrix (ECM) remodeling in 3D bioprinted scaffolds. (A) Bioprinted structures are initially (day 0) transparent but become opaque over time (day 60) due to cell-induced matrix remodeling and deposition of mineralized matrix. (B) Histology show progressive changes in the ECM of 3D bioprinted structures. Safranin O stains cartilage tissue in varying shades of red, while bone tissue is bluish-purple. Alcian Blue stains connective tissue light blue and cartilage dark blue. Together, these stains demonstrate the osteochondral production of cartilage ECM that transitions into mineralization. In osteochondral tissue formation, hMSCs differentiate into osteochondral progenitor cells and then into chondrocytes, producing a cartilaginous extracellular matrix. Chondrocytes then differentiate into preosteoblasts and direct the mineralization of the surrounding matrix. Copyright ACS Applied Materials and Interfaces
Extracellular matrix (ECM) remodeling in 3D bioprinted scaffolds. (A) Bioprinted structures are initially (day 0) transparent but become opaque over time (day 60) due to cell-induced matrix remodeling and deposition of mineralized matrix. (B) Histology show progressive changes in the ECM of 3D bioprinted structures. Safranin O stains cartilage tissue in varying shades of red, while bone tissue is bluish-purple. Alcian Blue stains connective tissue light blue and cartilage dark blue. Together, these stains demonstrate the osteochondral production of cartilage ECM that transitions into mineralization. In osteochondral tissue formation, hMSCs differentiate into osteochondral progenitor cells and then into chondrocytes, producing a cartilaginous extracellular matrix. Chondrocytes then differentiate into preosteoblasts and direct the mineralization of the surrounding matrix. Copyright ACS Applied Materials and Interfaces

Biocompatible heterogeneous bone incorporated with polymeric biocomposites for human bone repair by 3D printing technology

Authored by Meiling Wan  Shuifeng Liu  Da Huang  Yang Qu  Yang Hu  Qisheng Su  Wenxu Zheng  Xianming Dong  Hongwu Zhang  Yen Wei  Wuyi Zhou. Journal of Applied Polymer Science. 24 November 2020

Hemolysis test of composite materials. Copyright. Journal of Applied Polymer Science
Hemolysis test of composite materials. Copyright. Journal of Applied Polymer Science

Abstract:

Polylactic acid (PLA) has become a popular polymer material due to its superior biocompatibility. At present, there is little relevant research on heterogeneous bone powder. Besides, the poor dispersibility and adhesivity of inorganic particles in the organic phase remain a problem.

In this study, the pork bone powders were modified with N‐butanol to improve their dispersibility and compatibility in the PLA matrix. In addition, polybutylene succinate‐co‐terephthalates (PBSA) was applied as the flexibility to further reinforce the mechanical properties of materials. The composite filaments with a diameter of 1.75 ± 0.05 mm containing 10 wt% of modified bone powder, 10 wt% PBSA and 80 wt% PLA were prepared by a melt blending method.

The obtained results showed that modified particles were uniformly dispersed within the PLA matrix and improved the mechanical properties of the composite filaments with a tensile strength of 48.5 ± 0.2 MPa and bending strength of 79.1 ± 0.1 MPa and a notch impact strength of 15.8 ± 0.3 kJ/m2.

And the prepared composite materials contained low cytotoxicity, high biocompatibility, and printability, which verified the feasibility of it in 3D printing personalized bone repair applications. This provides a theoretical basis for further research on the effect of bone repair in vivo. Therefore, the composite material will have potential applications such as making customized bones and bone scaffolds by three-dimensional printing technology.

Extrusion-based 3D printed biodegradable porous iron

Authored by N.E. Putra, M.A. Leeflang, M. Minneboo, P. Taheri, L.E. Fratila-Apachitei, J.M.C. Mol, J. Zhou, A.A. Zadpoor. Acta Biomaterialia. May 2020

The starting material, extrusion-based 3D printing, and scaffold design: (a) iron powder particle morphology, (b) an illustration of extrusion-based 3D printing, and (c) the scaffold with the 0° and 90° lay-down pattern design. Copyright. Acta Biomaterialia
The starting material, extrusion-based 3D printing, and scaffold design: (a) iron powder particle morphology, (b) an illustration of extrusion-based 3D printing, and (c) the scaffold with the 0° and 90° lay-down pattern design. Copyright. Acta Biomaterialia

Abstract: 

Extrusion-based 3D printing followed by de-binding and sintering is a powerful approach that allows for the fabrication of porous scaffolds from materials (or material combinations) that are otherwise very challenging to process using other additive manufacturing techniques.

Iron is one of the materials that have been recently shown to be amenable to processing using this approach. Indeed, a fully interconnected porous design has the potential of resolving the fundamental issue regarding bulk iron, namely a very low rate of biodegradation.

However, no extensive evaluation of the biodegradation behavior and properties of porous iron scaffolds made by extrusion-based 3D printing has been reported.

Therefore, the in vitro biodegradation behavior, electrochemical response, the evolution of mechanical properties along with biodegradation, and responses of an osteoblastic cell line to the 3D printed iron scaffolds were studied.

An ink formulation, as well as matching 3D printing, de-binding, and sintering conditions, was developed to create iron scaffolds with a porosity of 67%, pore interconnectivity of 96%, and a strut density of 89% after sintering. X-ray diffractometry confirmed the presence of the α-iron phase in the scaffolds without any residuals from the rest of the ink.

Owing to the presence of geometrically designed macropores and random micropores in the struts, the in vitro corrosion rate of the scaffolds was much improved as compared to the bulk counterpart, with 7% mass loss after 28 days. The mechanical properties of the scaffolds remained in the range of those of trabecular bone despite 28 days of in vitro biodegradation.

The direct culture of MC3T3-E1 preosteoblasts on the scaffolds led to a substantial reduction in living cell count, caused by a high concentration of iron ions, as revealed by the indirect assays. On the other hand, the ability of the cells to spread and form filopodia indicated the cytocompatibility of the corrosion products. Taken together, this study shows the great potential of extrusion-based 3D printed porous iron to be further developed as a biodegradable bone substituting biomaterial.

Morphology and phase composition of the porous iron scaffolds: SEM images of (a, b, c) the as-printed iron scaffolds and (d, e, f) the as-sintered iron scaffolds at different magnifications, (g) the cross-section of the polished struts, and (h) the XRD pattern of the scaffolds after sintering. Copyright. Acta Biomaterialia
Morphology and phase composition of the porous iron scaffolds: SEM images of (a, b, c) the as-printed iron scaffolds and (d, e, f) the as-sintered iron scaffolds at different magnifications, (g) the cross-section of the polished struts, and (h) the XRD pattern of the scaffolds after sintering. Copyright. Acta Biomaterialia

3D Printing In Orthopedics: Implants, Drug Delivery, Bone Regeneration

Bone Grafts: Inducing Bone Regeneration with 3D Printed Porosity

Bio Fabrication Techniques for Bone and Cartilage Tissue Regeneration

3D Bioprinting Bone – One Defect At A Time

An Introduction to Scaffolds for Tissue Engineering of the Bone and Cartilage

Comments