Development of liposomal amniotic mesenchymal stem cell metabolite products (AMSC-MP) loaded-scaffold for in vitro osteogenesis induction.

The use of scaffolds with good biocompatibility and mechanical properties largely determines the success of bone defect therapy intended to improve quality-adjusted life years. Furthermore, amniotic mesenchymal stem cell metabolite product (AMSC-MP) is required to accelerate bone regeneration with sustained release required to maximize the effectiveness of therapy. This study aims to evaluate the effect of different phospholipids on the osteogenesis capacity of AMSC-MP liposome loaded into scaffold. The thin layer hydration method was employed to create the AMSC-MP liposomes which had different lipid components: L-α-phosphatidylcholine (PC) an unsaturated or hydrogenated soybean phosphatidylcholine (HSPC), a saturated phospholipid, with different types of charged phospholipids i.e., 1,2-dioleoyltrimethylammoniumpropane (DOTAP) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), which were cationic and non-ionic lipids respectively.
AMSC-MP liposomes were embedded in the scaffold during a 24-hour incubation period. The results indicated that the particle sizes of liposomes prepared with saturated phospholipids, HS-DOTAP (132.35 ± 2.75 nm) and HS-DOTAP (152.85 ± 5.86 nm) were larger than those with saturated phospholipids, namely PC-DOTAP (139.355 ± 2.75 nm) and PC-DOTAP (91.85 ± 0.07 nm). The use of DOTAP in AMSC-liposomes resulted in positively charged vesicles i.e., +5 mV for HSPC-DOTAP and +10 mV for PC-DOTAP. Moreover, the results of an MTT viability test conducted on 7f2 cells and MSC indicated that all the formulas demonstrate good in vitro biocompatibility. Formulations containing DOPE produced high cellular uptake values and had superior sustain release properties compared to the others. This result correlates positively with cell attachment to the liposome scaffold which was higher than the others. Moreover, DOPE-containing formulations increased RUNX2 expression, indicating enhanced in vitro osteogenesis capacity. Therefore, Liposome AMSC-MP loaded scaffold demonstrates high potential for use in bone regeneration therapy.
Graphical abstract
The AMSC-MP liposome-loaded scaffold allows the controlled release of AMSC-MP due to its encapsulation inside the liposome. However, burst release can also occur due to free AMSC-MP attached to the outer surface of the liposomes and the scaffold. Adding DOPE to AMSC-MP liposome results in sustained release properties that enhance cellular uptake. In addition, a positive correlation between the sustained release effect and cellular uptake improves cell attachment to the scaffold and osteogenesis activity based on enhancement of the RUNX2 gene, alkaline phosphatase, and calcium expression analysis.