Activating host macrophages with glucomannan-coated scaffold accelerates osteogenic differentiation

Objectives: This study investigates whether an acetyl-coated Bletilla striata polysaccharide (acBSP) scaffold system, loaded with adipose-derived mesenchymal stem cells (AD-MSCs), can activate host macrophages toward a pro-osteogenic phenotype and enhance osteogenic differentiation. Methods: To evaluate macrophage response to bisphosphonate (BP) treatment in vivo, Tg (mfap4:tdTomato) zebrafish were administered 100 µg/mL of BP weekly. After the 3rd, 6th, and 10th treatment cycles, fish were euthanized, and pharyngeal teeth were dissected and imaged. Differences in mean fluorescence intensity and macrophage numbers between BP-treated and untreated groups (n = 6 per group) were analyzed using ANOVA with Sidak’s post-test. In vitro, THP-1 cells were differentiated into mature macrophages and exposed to BPs for 24 hours. AD-MSCs were then cultured on acBSP-coated scaffolds and incubated with conditioned media (CM) from BP-treated macrophages. ELISA and RT-PCR were used to assess pro-inflammatory cytokines and osteogenic markers, evaluating the scaffold’s role in osteogenic differentiation. Results: In vivo analysis revealed significantly elevated mfap4 expression at 10 BP cycles (p < 0.0001), with no significant differences between cycles 3 and 6. Cryosection analysis demonstrated a marked increase in macrophage numbers (p < 0.0001) in BP-treated groups compared with controls. In vitro, THP-1 macrophages exhibited high viability on acBSP scaffolds. BP treatment reduced pro-inflammatory cytokine expression, while CM from BP-treated macrophages cultured on acBSP scaffolds significantly upregulated alkaline phosphatase (ALP) within seven days and osteocalcin (OCN) expression at later stages, indicating accelerated osteogenic differentiation. Conclusions: BP treatment enhances macrophage accumulation, and subsequent activation by acBSP scaffolds promotes osteogenic differentiation of AD-MSCs. This macrophage–scaffold interplay may offer a promising strategy for bone tissue engineering and regenerative therapies.