Nanoengineered Osteoinductive Bioink for 3D Bioprinting Bone Tissue

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 bioinks 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 osteoinducing 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 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 development of this NICE bioink technology toward a realistic clinical process for 3D bioprinting patient-specific bone tissue for regenerative medicine.

Keywords: bone bioprinting; hydrogels; ionic-covalent reinforcement; nanomaterials; osteoinductive bioinks.

Figure 1.

NICE bioink design and printability assessment. (A) The combination of gelatin methacrylate (GelMA), kappa-carrageenan (kCA), and nanosilicates (nSi) forms nanoengineered ionic-covalent entanglement (NICE) bioink, which demonstrates superior printability and mechanical properties compared to individual components. (B) Different compositions were tested to create a bioink that balanced the need for mechanical strength and osteogenic environment with a low polymer content, highly hydrated, remodelable environment. (C) The 3D printability of each bioink formulation was quantified using screw-driven extrusion printing of a warmed (37°C) bioink solution to create a 3cm tall, 1cm wide hollow tube. (D) Print success was based on height reached, conformity to expected dimensions, and lack of observable errors.

Figure 2.

Rheology of NICE bioink and mechanical performance of 3D printing structures. (A) Shear recovery tests showed that print performance corresponds well with rapid viscosity recovery, which reaches over 100% recovery due to thermal gelation. (B) NICE printed structures (7.5% GelMa, 1% kCA, 2% nanosilicates) are highly flexible and resilient. 3D printed tube structures (3 cm in height) can be completely collapsed and quickly regain their shape. (C) Mechanical testing showed that all 3D printed structures (100% infill density) were stiff to direct the osteogenic differentiation of encapsulated hMSC cells. The increasing concentration of polymer and nanoparticles improves mechanical stiffness and toughness. (D) NICE bioink can be printed into custom scaffolds or can be injected into the defect site.

Figure 3.

3D Printed NICE scaffolds support cell-induced matrix remodeling. (A) hMSCs are encapsulated in the NICE bioink and cell-laden scaffolds are printed (n=5). Initially the scaffolds are transparent but when cultured over 60 days, the scaffolds turn translucent due to remodeling and deposition of nascent proteins. The matrix remodeling in presence of cells is monitored by determining (B) mechanical stability and (C) scaffold mass in PBS, media and collagenase. 3D printed scaffolds completely degrade in PBS and media within 4 weeks. The presence of collagenase degrades the scaffolds within 2 weeks. For bioprinted scaffolds (loaded with hMSCs), no significant mass loss was observed even after 100 days of cultures. This indicates that matrix deposition by cells is able to keep the structure intact. (D) SEM images taken at different times show gradual changes in the microstructure of cell-containing 3D bioprinted scaffolds. Over time, a decrease in pore size was observed due to deposition of ECM by cells.

Figure 4.

Extracellular matrix remodeling in 3D bioprinted scaffolds. (A) Bioprinted structures are initially transparent but become opaque over time due to cell-induced matrix remodeling and deposition of mineralized matrix. (B) Histology images show progressive changes in the ECM of 3D bioprinted structures. Safranin O stains cartilage tissue 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.

Figure 5.

Mineralized matrix in 3D bioprinted scaffolds. (A) Von Kossa and Alizarin Red staining reveals mineralization with calcium, carbonates, and phosphates. (B) A calcium-cresolphthalein complexone assay quantifies calcium content in dried gels over time. (C) SEM-EDS imaging visualizes the increase in calcium content over time. (D) EDS quantitative data shows a concurrent increase in calcium and phosphates, as would be expected with osteogenic tissue formation.

Figure 6.

Changes in hMSC gene expression in response to nanosilicates after 21 days of culture. (A) We can investigate the effects of nanosilicates on gene expression profile by subjecting hMSCs to nanosilicates (in absence of any growth factors) for 21 days. hMSCs differentiate down an osteochondral pathway by SOX9 gene expression, and interplay between morphogenetic signaling molecules (including TGF-β and BMP) mediates between chondrogenic and osteogenic cell behavior. (B) High correlation between two replicates for hMSCs control and hMSCs treated with nanosilicates (hMSCs_nSi). (C) Differential gene expression analysis releveled significant number of gene are either up (red) or down (blue) regulated due to nanosilicates treatment. (D) Gene ontology analysis revel multiple signaling pathway triggered by nanosilicates. (E) Selected differentially regulated genes (due to nanosilicate treatment) that play important role in endochondral differentiation are highlighted. (F) Gene tracks of SMAD1, SOX9 and TGFBR2 showing effect of nanosilicate on hMSCs.