Nanosecond pulsed electric fields prime mesenchymal stem cells to peptide ghrelin and enhance chondrogenesis and osteochondral defect repair in vivo

Mesenchymal stem cells(MSCs) are important cell sources in cartilage tissue development and homeostasis,and multiple strategies have been developed to improve MSCs chondrogenic differentiation with an aim of promoting cartilage regeneration.Here we report the effects of combining nanosecond pulsed electric fields(ns PEFs) followed by treatment with ghrelin(a hormone that stimulates release of growth hormone) to regulate chondrogenesis of MSCs.ns PEFs and ghrelin were observed to separately enhance the chondrogenesis of MSCs,and the effects were significantly enhanced when the bioelectric stimulation and hormone were combined,which in turn improved osteochondral tissue repair of these cells within Sprague Dawley rats.We further found that ns PEFs can prime MSCs to be more receptive to subsequent stimuli of differentiation by upregulated Oct4/Nanog and activated JNK signaling pathway.Ghrelin initiated chondrogenic differentiation by activation of ERK1/2 signaling pathway,and RNA-seq results indicated 243 genes were regulated,and JAK-STAT signaling pathway was involved.Interestingly,the sequential order of applying these two stimuli is critical,with ns PEFs pretreatment followed by ghrelin enhanced chondrogenesis of MSCs in vitro and subsequent cartilage regeneration in vivo,but not vice versa.This synergistic prochondrogenic effects provide us new insights and strategies for future cell-based therapies.

In situ self-assembled organoid for osteochondral tissue regeneration with dual functional units

The regeneration of hierarchical osteochondral units is challenging due to difficulties in inducing spatial,directional and controllable differentiation of mesenchymal stem cells(MSCs)into cartilage and bone compartments.Emerging organoid technology offers new opportunities for osteochondral regeneration.In this study,we developed gelatin-based microcryogels customized using hyaluronic acid(HA)and hydroxyapatite(HYP),respectively for inducing cartilage and bone regeneration(denoted as CH-Microcryogels and OS-Microcryogels)through in vivo self-assembly into osteochondral organoids.The customized microcryogels showed good cytocompatibility and induced chondrogenic and osteogenic differentiation of MSCs,while also demonstrating the ability to self-assemble into osteochondral organoids with no delamination in the biphasic cartilage-bone structure.Analysis by mRNA-seq showed that CH-Microcryogels promoted chondrogenic differentiation and inhibited inflammation,while OS-Microcryogels facilitated osteogenic differentiation and suppressed the immune response,by regulating specific signaling pathways.Finally,the in vivo engraftment of pre-differentiated customized microcryogels into canine osteochondral defects resulted in the spontaneous assembly of an osteochondral unit,inducing simultaneous regeneration of both articular cartilage and subchondral bone.In conclusion,this novel approach for generating self-assembling osteochondral organoids utilizing tailor-made microcryogels presents a highly promising avenue for advancing the field of tissue engineering.