Congenital abnormalities, trauma, and disease result in significant demands for bone replacement in the craniofacial region and across the body. Tetra-compositions of organic and inorganic scaffolds could provide advantages for bone regeneration. This research aimed to fabricate and characterize amorphous calcium phosphate (ACP)/calcium sulfate hemihydrate (CSH) with alginate/cellulose composite scaffolds using 3D printing. Alginate/cellulose gels were incorporated with 0%, 13%, 15%, 18%, 20%, and 23% ACP/CSH using the one-pot process to improve morphological, physiochemical, mechanical, and biological properties. SEM displayed multi-staggered filament layers with mean pore sizes from 298 to 377 μm. A profilometer revealed mean surface roughness values from 43 to 62 nm that were not statistically different. A universal test machine displayed the highest compressive strength and modulus with a statistical significance in the 20% CP/CS group. FTIR spectroscopy showed peaks in carbonate, phosphate, and sulfate groups that increased as more ACP/CSH was added. Zero percent of ACP/CSH showed the highest swelling and lowest remaining weight after degradation. The 23% ACP/CSH groups cracked after 60 days. In vitro biocompatibility testing used the mouse osteoblast-like cell line MC3T3-E1. The 18% and 20% ACP/CSH groups showed the highest cell proliferation on days five and seven. The 20% ACP/CSH was most suitable for bone cell regeneration.

New bone formation starts from the initial reaction between a scaffold surface and the extracellular matrix. This research aimed to evaluate the effects of various amounts of calcium, phosphate, sodium, sulfur, and chloride ions on osteoblast-like cell differentiation using tetra-polymers of amorphous calcium phosphate (ACP), calcium sulfate hemihydrate (CSH), alginic acid, and hydroxypropyl methylcellulose. Moreover, 3D-printed scaffolds were fabricated to determine the ion distribution and cell differentiation. Various proportions of ACP/CSH were prepared in ratios of 0%, 13%, 15%, 18%, 20%, and 23%. SEM was used to observe the morphology, cell spreading, and ion complements. The scaffolds were also examined for calcium ion release. The mouse osteoblast-like cell line MC3T3-E1 was cultured to monitor the osteogenic differentiation, alkaline phosphatase (ALP) activity, total protein synthesis, osteocalcin expression (OCN), and calcium deposition. All 3D-printed scaffolds exhibited staggered filaments, except for the 0% group. The amounts of calcium, phosphate, sodium, and sulfur ions increased as the amounts of ACP/CSH increased. The 18%ACP/CSH group significantly exhibited the most ALP on days 7, 14, and 21, and the most OCN on days 14 and 21. Moreover, calcium deposition and mineralization showed the highest peak after 7 days. In conclusion, the 18%ACP/CSH group is capable of promoting osteoblast-like cell differentiation on 3D-printed scaffolds.