Fibroin/sulfated alginate membranes containing exosomes of stem cells treated with B and Zn-doped hydroxyapatite for periosteal tissue engineering.

Periosteal regeneration requires tissue engineering strategies that simultaneously support osteogenesis, angiogenesis, and immunomodulation. In this study, fibroin/sulfated alginate (F/sA) composite membranes loaded with exosomes derived from human adipose-derived stem cells (hADSCs) treated with pure, 4 mol% B-doped, and 4 mol% B and 8 mol% Zn-doped hydroxyapatite (HA) were developed for periosteal tissue engineering. It was hypothesized that incorporation of sA and HA-conditioned exosomes enhances osteoimmunomodulation while preserving scaffold integrity. F/sA membranes with varying sulfated alginate content (0-20 wt%) were fabricated and characterized. Increasing sulfated alginate content enhanced water uptake and degradation while reducing tensile strength and Young's modulus. A 95 : 5 F/sA ratio was identified as optimal exosome delivery composition, exhibiting the highest hADSC viability and reducing IL1B and CASP1 levels in THP-1 macrophages (p < 0.05). It was also found that exosomes isolated from pure and doped HA-treated hADSCs showed treatment-dependent alterations in the cargo. The 8 mol% B-doped HA group significantly increased exosome yield, while all HA treatments reduced protein/particle and DNA/particle ratios. Doped HA treatments significantly increased RNA/particle ratio (p < 0.05). Exosome-loaded F/sA membranes enhanced early cell attachment and proliferation and significantly promoted osteogenic differentiation of hADSCs, increasing RUNX2, COL1A1, and OCN expression. Immunomodulatory effects were observed for all exosome groups, except for the 4 mol% B and 8 mol% Zn-doped HA groups, which significantly increased CASP1 expression. Overall, this study demonstrates that HA doping modulates exosomal composition in a dopant-dependent manner and that exosome-functionalized F/sA membranes constitute a multifunctional periosteal substitute with tunable properties.