Hybrid 3D Bioprinting Technology: Biocompatibility Assessment and Mechanical Characterization of Fabricated Constructs

Introduction: Bioprinting, an emerging biomedical technology, shows great potential with its applications in bioengineering, tissue engineering and regenerative medicine. Development and application of 3D bioprinting is of critical importance in managing musculoskeletal disorders, a significant health concern that resonates with the growing aging demographic and an estimated 1.71 billion individuals globally affected by these medical conditions. Although significant advancements have been achieved in bioprinting for musculoskeletal applications, the integration of hard and soft multifunctional components into a cohesive, tissue-like structure remains a complex endeavor yet to be achieved. This study discusses the technology of a 3D Hybrid Bioprinter (Hybprinter) which is particularly developed for regenerative tissue engineering through the creation of 3D composite constructs where both hard and soft tissues are involved, as well as hard-soft interfacial tissues. The Hybprinter technology is meticulously developed by combining two bioprinting techniques into a singular 3D bioprinter capable of fabricating hybrid constructs with integrated soft and hard tissue engineered components. We have shown that the Hybprinter facilitates a seamless integration and customization of complex tissue configurations with biocompatible soft and hard components. In this approach, we study the optimal bioprinting conditions and assess cell-laden bioink characteristics that are compatible with Hybprinter technology. Our investigation centers on effectively resolving the fundamental balance between biocompatibility and printability, specifically tailored for the needs of musculoskeletal tissue engineering applications.

Materials and

Methods: The Hybprinter technology combines two different bioprinting systems, namely Fused Deposition Modeling (FDM) and Digital Light Processing-based Stereolithography (DLP-SLA) to seamlessly fabricate and integrate 3D rigid and soft components (Fig.1A). The Hybprinter operates using a layer-by-layer approach, with the FDM system fabricates robust porous polymer/ceramic scaffolds for structural reinforcement, while SLA system fabricates soft hydrogel components laden with bioactive elements (Fig.1B,C). In this study the Hybprinter is used to fabricate hybrid contracts and cell-laden hydrogel samples. For fabrication of the rigid parts with FDM, a distinct thermoplastic material is used that constitutes a composite of polycaprolactone (PCL) and β-tricalcium phosphate (β-TCP) which provides a suitable balance between printability, biodegradation, and tissue regeneration. Fabrication of soft hydrogel parts within hybrid constructs is accomplished through visible light projection on photo-crosslinkable bioink. Our bioink formulation composed of 10% w/v GelMA hydrogel and a density of 1×10^6 cells/mL of human bone marrow-derived mesenchymal stem cells (hMSCs), mixed with lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator and Tartrazine photo-absorber. Mechanical properties of the fabricated hybrid construct and hydrogel samples are investigated through a series of compression tests and followed by studying the correlation between the light exposure time and hydrogel stiffness (Fig.1E). Biocompatibility of the bioink is investigated through live-dead viability assays, accompanied by Fluorescent imaging (Fig.1F,G). Lastly, optimal printing parameters and hydrogel properties are investigated using both mechanical test results and cellular viability experiments. This approach serves to define optimal print conditions and characterize an optimal Gelma bioink that effectively provides a balance between biocompatibility and printability.

Results, Conclusions, and Discussions: This study discusses the 3D Hybprinter technology for bioprinting hybrid constructs containing hard robust scaffolds and soft bioactive hydrogels for bone and musculoskeletal tissue regeneration. In Hybprinter, the FDM system 3D prints solid segments using biodegradable PCL/β-TCP material and the DLP-SLA system 3D prints integrated soft hydrogel segments of the hybrid construct. The performance of the Hybprinter is first demonstrated by 3D printing a hybrid construct, comprised of 90% hydrogel and 10% hard scaffold. Mechanical tests conducted on the resulting hybrid construct display a compressive stiffness in the region of 5 MPa which represents a significant improvement compared to hydrogel constructs, which typically lie within the kPa order (Fig.1D). These results underline the crucial potential role of hybrid bioprinting in musculoskeletal applications, where equal emphasis is placed on mechanical strength and biocompatibility. Subsequently, a photo-crosslinkable GelMA bioink is devised with the intention of fabricating cell-laden hydrogel samples to investigate biocompatibility and printability. Mechanical compression tests conducted on the hydrogel samples demonstrate attainable compressive modulus in the scope of 10-25 kPa, which can be accomplished via controlling the bioprint conditions. Lastly, an optimal combination of gel composition and bioprinting conditions is obtained through a set of concurrent live-dead viability and mechanical testing. The results show that, at the optimal printing condition the viability consistently remains above 90% at both day 1, day 3, day 5, and day 7 (Fig.1F,G). Concurrently, the optimum printing conditions accomplish the printability requirements, leading to the attainment of a compressive modulus of 14.0±0.4 kPa which is suitable for hMCS growth (Fig.1E). The representative fluorescent Live-Dead stained image of a hMSC-laden sample printed under optimal conditions clearly shows that the hMSCs become elongated, spread out, and connect with each other within the 3D printed GelMA hydrogel after 7 days of incubation. Results obtained from this preliminary study propose diverse potential applications for the Hybprinter in the field of tissue engineering where the solid segment provides mechanical support and the soft hydrogel segment contributes to biological efficacy of the hybrid construct, acting as a vehicle for the delivery of bioactive entities such as cells, drugs, and growth factors.

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