Objectives: Three-dimensional (3D) bioprinting is a manufacturing process that incorporates viable cells into a 3D matrix by adding layer upon layer of material. The objectives of this study are to characterize a novel matrix of collagen and hydroxyapatite and to assess the effects of the 3D bioprinting process on cytotoxicity, proliferation rate, and cytokine expression of Homo sapiens palatal mesenchyme (HEPM) cells. Methods: For this, we prepared a 3D matrix of collagen and hydroxyapatite without and with cells. We used light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) to characterize the structure and arrangement of the collagen fibers. We then incubated the matrix with known standards of cytokines to measure adsorption. We assessed the proliferation and viability of HEPM cells in the presence of the 3D construct and after 3D bioprinting. Finally, we assessed the cytotoxicity of this matrix for HEPM cells and assessed its effect on the production of chemokines and cytokines. A one-way fixed effect ANOVA was fit to concentrations of cytokines and pairwise group comparisons were conducted using Tukey’s Honest Significant Differences test (p< 0.05). Results: The matrix was found to contain interwoven strands of collagen and some hydroxyapatite crystals that did not absorb cytokines except for MIP-1a (p< 0.05). The matrix was found to be non-cytotoxic using an AlamarBlue® assay. We found that the cell proliferation rate was lower when seeded on the 3D construct than in 2D culture. We also found that the proliferation rate was very low for the HEPM cells in the 3D bioprinted constructs. In the presence of the 3D construct, the HEPM cells had similar expression profiles of the cytokines measured (P > 0.05 for GM-CSF, IL-6, IL-8, and RANTES). Conclusion: 3D-bioprinting has the potential to be used in dentistry as a novel osteogenic bone grafting material. Here we show that a novel matrix of collagen and hydroxyapatite is non-cytotoxic to HEPM cells and does not induce a proinflammatory response.