Jagged1 immobilization to an osteoconductive polymer activates the Notch signaling pathway and induces osteogenesis.

Treatment of nonunion fractures is a significant problem. Common therapeutics, including autologous bone grafts and bone morphogenetic proteins, show well-established limitations. Therefore, a need persists for the identification of novel clinical therapies to promote healing. The Notch signaling pathway regulates bone development. Clinically, loss-of-function mutations to the Notch ligand Jagged1 decrease bone mass and increase fracture risk. Jagged1 is also the most highly upregulated ligand during fracture repair, identifying it as a potential target to promote bone formation. Therefore, the objective of this study was to develop a clinically translatable construct comprised of Jagged1 and an osteoconductive scaffold, and characterize its activity in human mesenchymal stem cells (hMSC). We first evaluated the effects of Jagged1 directly immobilized to a novel poly(β-amino ester) relative to indirect coupling via antibody. Direct was more effective at activating hMSC Notch target gene expression and osteogenic activity. We then found that directly immobilized Jagged1 constructs induced osteoblast differentiation. This is the first study to demonstrate that Jagged1 delivery transiently activates Notch signaling and increases osteogenesis. A positive correlation was found between Jagged1-induced Notch and osteogenic expression. Collectively, these results indicate that Jagged1 coupled to an osteogenic biomaterial could promote bone tissue formation during fracture healing.

Dishowitz MI ，Zhu F ，Sundararaghavan HG ，Ifkovits JL ，Burdick JA ，Hankenson KD ... -  《-》

Jagged1 promotes mineralization in human bone-derived cells.

The present study aimed to investigate the expression of Notch signaling components during osteogenic differentiation in vitro and bone healing in vivo. In addition, the influence of Notch signaling on osteogenic differentiation of human bone-derived cells was examined. Gene expression profiling of osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells in vitro (GSE80614) and bone healing period of murine tibial fracture in vivo (GSE99388) was downloaded from Gene Expression Omnibus database. The expression of Notch signaling components was obtained from bioinformatic tools. Human bone-derived cells were isolated from alveolar and iliac bone. Cells were seeded on Jagged1 immobilized surface. Osteogenic marker gene expression and mineralization were examined using real-time polymerase chain reaction and alizarin red s staining, respectively. From bioinformatic analysis of gene expression profiling, various Notch signaling components were differentially expressed during osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells in vitro and bone healing period of murine tibial fracture in vivo. The common genes differentially regulated of these two datasets were Hes1, Aph1a, Nsctn, Furin, Adam17, Hey1, Pcsk5, Nedd4, Jag1, Heyl, Notch3, Dlk1, and Hey2. For an in vitro analysis, the mineral deposition markedly increased after seeding human bone-derived cells on Jagged1 immobilized surface, correspondingly with the increase of ALP mRNA expression. Jagged1 treatment downregulated TWIST2 mRNA expression in both human alveolar and iliac bone-derived cells. Notch signaling is regulated during osteogenic differentiation and bone healing. In addition, the activation of Notch signaling promotes osteogenic differentiation in human alveolar and iliac bone-derived cells. Therefore, Notch signaling manipulation could be a useful approach for enhancing bone regeneration.

Osathanon T ，Manokawinchoke J ，Sa-Ard-Iam N ，Mahanonda R ，Pavasant P ，Suwanwela J ... -  《-》

PKCδ is required for Jagged-1 induction of human mesenchymal stem cell osteogenic differentiation.

JAG1, the gene for the Jagged-1 ligand (Jag1) in the Notch signaling pathway, is variably mutated in Alagille Syndrome (ALGS). ALGS patients have skeletal defects, and additionally JAG1 has been shown to be associated with low bone mass through genome-wide association studies. Plating human osteoblast precursors (human mesenchymal stem cells-hMSCs) on Jag1 is sufficient to induce osteoblast differentiation; however, exposure of mouse MSC (mMSC) to Jag1 actually inhibits osteoblastogenesis. Overexpression of the notch-2 intracellular domain (NICD2) is sufficient to mimic the effect of Jag1 on hMSC osteoblastogenesis, while blocking Notch signaling with a γ-secretase inhibitor or with dominant-negative mastermind inhibits Jag1-induced hMSC osteoblastogenesis. In pursuit of interacting signaling pathways, we discovered that treatment with a protein kinase C δ (PKCδ) inhibitor abrogates Jag1-induced hMSC osteoblastogenesis. Jag1 results in rapid PKCδ nuclear translocation and kinase activation. Furthermore, Jag1 stimulates the physical interaction of PKCδ with NICD. Collectively, these results suggest that Jag1 induces hMSC osteoblast differentiation through canonical Notch signaling and requires concomitant PKCδ signaling. This research also demonstrates potential deficiencies in using mouse models to study ALGS bone abnormalities.

Zhu F ，Sweetwyne MT ，Hankenson KD 《-》

Surface-bound orientated Jagged-1 enhances osteogenic differentiation of human periodontal ligament-derived mesenchymal stem cells.

Notch signaling plays critical roles in various cell types by regulating cell fate determination and differentiation. Here, we investigated the ability to control differentiation of human periodontal ligament derived mesenchymal stem cells using modified surfaces containing the affinity immobilized Notch ligand, Jagged-1. After seeding human periodontal ligament derived mesenchymal stem cells (HPDLs) on Jagged-1 modified surfaces, expression of Notch signaling target genes, Hes-1 and Hey-1, was higher than those exposed to soluble Jagged-1 or control surfaces. Upregulation of Notch signaling target genes was attenuated after treatment with the γ secretase inhibitor. Upon seeding the cells on Jagged-1 immobilized surface and maintained in osteogenic medium, alkaline phosphatase enzymatic activity and mineralization as well as mRNA expression of alkaline phosphatase (ALP), collagen type I (COL I) and osteopontin (OPN) were significantly increased compared to those of controls. However, osteocalcin (OCN) mRNA expression level was decreased when cells were exposed to Jagged-1 modified surfaces. HPDLs on Jagged-1 modified surfaces expressed lower TWIST2 mRNA levels than the control, suggesting that the mechanism whereby Jagged-1 enhances osteogenic differentiation of HPDLs may occur through Notch signaling and TWIST regulation. In summary, an alteration of biomaterial interface using Notch ligands illustrates a promising system to control HPDLs differentiation toward osteogenic lineage.

Osathanon T ，Ritprajak P ，Nowwarote N ，Manokawinchoke J ，Giachelli C ，Pavasant P ... -  《-》

Induction of myogenic differentiation of human mesenchymal stem cells cultured on Notch agonist (Jagged-1) modified biodegradable scaffold surface.

Wen F ，Wong HK ，Tay CY ，Yu H ，Li H ，Yu T ，Tijore A ，Boey FY ，Venkatraman SS ，Tan LP ... -  《-》