Exploring 3D Printed GelMA/Hydroxyapatite Scaffold from Design to Biological Evaluation for Bone Tissue Engineering

Bone repair is a widely explored field in tissue engineering due to its significant impact on the human body's structural integrity. Biomaterials based on calcium phosphate, such as hydroxyapatite (HAp), have shown promise because of their ability to emulate the mineral composition and porous structure of bone. Additionally, the utilization of additive manufacturing has provided opportunities to create cellular scaffolds for bone grafts, allowing us to control properties like porosity, pore size, structural form, and mechanical characteristics. These designs are crafted using Computer-Aided Design (CAD) software. The primary objective of this project is to design, fabricate, characterize, and assess the bioactivity of 3D HAp scaffolds for tissue engineering applications. To achieve this, hydroxyapatite was initially synthesized using the microwave-assisted hydrothermal method. Consequently, HAp nanofibers acquired a hexagonal structure, enhancing their mechanical properties. Subsequently, the HAp nanofibers underwent characterization through X-ray diffraction (XRD) and Scanning Electron Microscopy (SEM) to determine dimensions, morphology, topology, orientation, and crystalline structure. In addition, various 3D bone scaffolds with interconnected pores, facilitating permeability similar to trabecular bone morphology, were designed using Rhinoceros 3D CAD software with the Grasshopper extension. These scaffolds were produced using a 3D bioprinter with methacryloyl (GelMA) and laminin as bioink. Gelatin methacryloyl (GelMA) hybrid hydrogel with hydroxyapatite has shown its ability to promote osteogenesis and enhance osseointegration, as supported by both in vitro and in vivo studies. In this research, 3D Voronoi bioprinted scaffolds with GelMA/HAp were designed and manufactured for in vitro biological evaluation using mesenchymal stem cells. Mesenchymal Stem Cells obtained through primary culture of pig bone marrow were cultured in DMEM medium with 10% inactivated fetal bovine serum at 37°C/5% CO₂. The study included two groups: one with the GelMA/HAp scaffold and another with a control group containing only GelMA hydrogel without hydroxyapatite. The scaffolds were maintained for 21 days under these conditions. Then, Alizarin red staining was done to reveal the osteogenic differentiation of Mesenchymal Stem Cells. Subsequently, RNA was extracted from the cell suspension, and osteogenesis was evaluated through RT-qPCR with markers BMP-2, OCN, COL1A1, and OPN. The mean cycle threshold (Ct) value for each target gene was normalized against the Ct value of a housekeeping gene to determine relative expression. To calculate the fold change, the DDCt method was applied, comparing mRNA expressions between the treated groups (with GelMA/HAp) and the negative group (cells cultured in complete culture medium only). Based on robust scientific evidence, this biomaterial is expected to demonstrate its bioactive functions without harming the cells, as intended.