Abstract
Amniotic membrane (AM) is a tissue that forms the inner portion of the placenta and is known to contain anti-inflammatory and anti-scarring properties. Due to these properties, AM has been used in many clinical applications to downregulate the inflammation, reduce scar formation, and ultimately promote wound healing. One potential clinical unmet need where AM use could be potentially beneficial is bony non-unions, wherein skeletal bone fails to heal after 9 months of injury. Based on this need, we evaluated and demonstrated AM time- and dose-dependently promotes calcification of pre-osteoblasts (MC3T3-E1 cells), a known characteristic of osteogenesis. Such effect was independent of exogenous osteogenic supplements. We further showed HC-HA/PTX3 purified from AM promoted in vitro osteogenesis of human bone marrow mesenchymal stem cells (MSCs) through an endochondral ossification mediated pathway as demonstrated by activation of BMP signaling (a known promoter of the bone morphogenic process) and promotion of chondrogenic (collagen type II and aggrecan) and osteogenic (osterix, osteocalcin, and alkaline phosphatase) markers. Cellular aggregation was demonstrated to be an early morphological event needed to promote osteogenesis and was inhibited by AMD3100 and alpha CD44 blocking antibody suggesting CXCR4 and CD44 dependence. CXCR4 and CD44 were also shown to physically interact and form a heterodimer at 5 minutes after cell seeding which further suggests their interdependence. This interaction was followed by nuclear translocation of CXCR4 at 24 hours which was inhibited by AMD3100. We further show HC-HA/PTX3 can be reconstituted in vitro and such complex has a biochemical composition similar to HC-HA/PTX3 purified from AM with presence of HC1 and high molecular weight PTX3, but not TSG-6, HC2, and HC3. We further showed cell aggregation and subsequent endochondral ossification of MSCs can be recapitulated with reconstituted HC-HA/PTX3 but not HC1, HA, or PTX3 alone.