Influence of Ti-6Al-4V Scaffold Architecture on Early Cellular Responses Relevant to Bone Regeneration

Background: Optimal porosity and surface area of additively manufactured Ti-6Al-4V scaffolds for early human mesenchymal stem cell (hMSC) retention remain unclear, despite their importance in reconstruction of critical-sized bone defects. This study evaluated scaffold architectures fulfilling biomechanical criteria for hMSC and osteocyte survival, proliferation, differentiation, cell cycle, and retention. Methods: Primary hMSCs from four healthy donors were characterized by flow cytometry, immunofluorescence, and Western blotting. Cells were seeded onto Ti-6Al-4V scaffolds with graded porosity (P50-P90, 50-90% porosity) and cultured for 120 h. Viability and retention were measured by trypan blue exclusion, apoptosis and cell cycle by flow cytometry, and osteogenic differentiation by collagen I, osteocalcin, and Akt phosphorylation analyses. Collagen-embedded hMSCs and osteoblasts were used to assess migration and phenotype maintenance. Results: The densest scaffold, P50, consistently retained significantly more viable hMSCs and differentiated osteoblasts than P60 and P70 (p < 0.005 in both cases, One-way ANOVA analysis, significance level a = 0.05 followed by Bonferroni correction). Among the scaffold architectures investigated, P50 demonstrated the most favorable early cellular retention under the present experimental conditions. No scaffold-induced apoptosis or proliferation changes were detected. Osteogenic differentiation and Akt phosphorylation increased during culture. In collagen-containing scaffolds, cells migrated from the matrix and preferentially coated titanium struts, with P50 supporting superior attachment. Conclusions: Among the scaffold architectures investigated, the low-porosity/high-surface-area P50 design provided the most favorable early microenvironment for hMSC and osteoblast retention without cytotoxic effects.