Introduction: The effective treatment of severe bone defects remains a significant medical challenge, with synthetic bone substitutes being the primary approach for repair. Biomaterials that emulate the structural, mechanical, and biological characteristics of natural bone are widely used to address bone defects and promote in vivo bone regeneration. In tissue engineering applications, active scaffolds based on hydrogels have gained considerable interest due to their highly porous structure and flexibility, which mimic the extracellular matrix for cell growth. Consequently, 3D bioprinting techniques are popular for creating scaffolds with diverse shapes and dimensions. Gelatin methacryloyl (GelMA), a modified form of gelatin using methacrylic anhydride, serves as a foundation for 3D-printable hydrogel bioinks. GelMA can be covalently crosslinked under UV light in the presence of a photoinitiator, forming 3D structures with customizable geometry and adjustable mechanical properties suitable for various scaffold requirements. These hydrogels demonstrate numerous beneficial biological features and are being explored for applications ranging from drug delivery to tissue engineering. GelMA's inherent Arg-Gly-Asp (RGD) sequences enhance biological interactions between cells and scaffolds. Significant advancements have shown that scaffold materials containing ions possess superior bone regeneration capabilities due to their ability to chemically bond with bone tissue in vivo. Notably, ions play a crucial role in regulating various cellular functions and enzymes, significantly impacting cellular homeostasis. Recent research has focused on incorporating bioactive ions into bone substitutes to stimulate vascularized bone repair.
The objective of this review is to summarize the ways inorganic ions influence tissue regeneration and to provide an overview of studies on 3D-printed scaffolds that incorporate bioactive ions
Methods: To create methacrylate gelatin, the initial phase involved dissolving gelatin in PBS (pH=7.4) at 50 ◦C. Following complete gelatin solubilization in PBS (approximately 45 minutes), methacrylic anhydride was introduced gradually. This substance interacted with the gelatin structure's functional groups for roughly 2.5 hours under constant magnetic stirring at 50 ◦C. The final stage consisted of dialyzing the methacrylate gelatin to eliminate any remaining methacrylic anhydride. This dialysis process was conducted at 40 ◦C for 5 days, utilizing cellulose dialysis bags and distilled water. The resulting GelMA underwent lyophilization at 0.025 bars for 24 hours to dry it.
The composite materials were prepared using the following method: Initially, the optimal concentration of selected ions from the total GelMA amount was dispersed in PBS for 1 hour and then sonicated for 5 minutes. Subsequently, GelMA was introduced and allowed to react for 24 hours at 40 ◦C. Once a uniform mixture was achieved, Irgacure was added at 1% w/v concentration relative to the total GelMA amount and dissolved through gentle stirring at 40 ◦C. The resulting bio-ink was then loaded into the 3D printing cartridge. All samples underwent biological characterization to determine the most cell-compatible ion concentration.
Results: Recent findings demonstrate that the newly created bio-inks, composed of 25%GelMA annorganic filler and 30%GelMA-annorganic filler, enabled the production of scaffolds with specific architectural designs. These structures could potentially serve as an appropriate environment for bone regeneration, as they promote cell attachment, expansion, and multiplication. The research indicates that ions like Zn2+ and Mg2+ have a beneficial impact on the process of osteogenic differentiation. The GelMA/ions scaffolds exhibited the ability to synergistically enhance in situ bone regeneration by increasing osteogenesis and stimulating endothelial cell functions. These results highlight the positive influence of ions on mechanical performance while preserving good cytocompatibility and the ability to support Bone marrow stromal cells (BMSCs) in their adhesion, proliferation, and osteogenic differentiation. The expression of osteopontin (OPN) and osterix (OSX) genes was verified at the protein level through immunofluorescence techniques combined with confocal microscopy.
Conclusion: The enhanced 3D bioprinted framework, created by integrating inorganic ions into bioink, improves tissue regeneration and mimics natural biological formations.
The main objective of this study was to examine 3D printable hydrogels based on GelMA/ions with potential applications and to investigate the effect of GelMA/ions on bone regeneration.
Keywords: 3D Printing- GelMA- Bone regeneration -Osteogenesis- Ions