Nonmineralized and Mineralized Collagen Scaffolds Induce Differential Osteogenic Signaling Pathways in Human Mesenchymal Stem Cells

Abstract

The instructive capabilities of extracellular matrix components in progenitor cell differentiation have recently generated significant interest in the development of bioinspired materials for regenerative applications. Previously, a correlation was described between the osteogenic capabilities of nanoparticulate mineralized collagen glycosaminoglycan scaffolds (MC-GAG) and an autogenous activation of small mothers against decapentaplegic ( Smad1/5) in the canonical bone morphogenetic protein receptor (BMPR) pathway with a diminished extracellular signal regulated kinase 1/2 (ERK1/2) activation when compared to nonmineralized collagen glycosaminoglycan scaffolds (Col-GAG). This work utilizes a canonical BMPR inhibitor (dorsomorphin homologue 1, DMH1) and an inhibitor of the mitogen activated protein kinase/ERK kinase (MEK)/(ERK) cascade (PD98059) to characterize the necessity of each pathway for osteogenesis. While DMH1 inhibits runt-related transcription factor 2 (Runx2) and bone sialoprotein II (BSPII) gene expression of primary human mesenchymal stem cells (hMSCs) on MC-GAG, PD98059 inhibits BSPII expression on Col-GAG independent of Runx2 expression. DMH1 inhibits mineralization on both Col-GAG and MC-GAG, however, PD98059 only inhibits mineralization on Col-GAG. DMH1 inhibits both Smad1/5 phosphorylation and Runx2 protein expression, whereas PD98059 inhibits ERK1/2 and c-Jun amino-terminal kinase 1/2 (JNK1/2) phosphorylation without affecting Runx2. Thus, activation of the canonical BMPR signaling is necessary for osteogenic differentiation and mineralization of hMSCs on Col-GAG or MC-GAG. The MEK/ERK cascade, intimately tied to JNK activation, is necessary for Runx2-independent osteogenesis on Col-GAG, while completely dispensable in osteogenesis on MC-GAG.

Nanoparticulate mineralized collagen glycosaminoglycan materials directly and indirectly inhibit osteoclastogenesis and osteoclast activation

“…We previously reported differential regulation of osteogenic differentiation and mineralization of primary hMSCs on Col‐GAG and MC‐GAG (Ren, Bischoff, et al, ; Ren, Weisgerber, et al, ; Zhou et al, ). MC‐GAG demonstrated an autogenous activation of the BMPR signalling in hMSCs that greatly surpasses Col‐GAG.…”

“…We have previously demonstrated the capabilities of MC‐GAG to induce in vitro hMSC osteogenic differentiation and mineralization as well as in vivo bone healing beyond that of a nonmineralized Col‐GAG control material (Lee et al, ; Ren, Tu, et al, ; Ren, Weisgerber, et al, ; Ren, Bischoff, et al, ; Weisgerber, Caliari, & Harley, ; Zhou et al, ). To evaluate the role of MC‐GAG in the regulation of osteoclast activation during osteoprogenitor differentiation, bone marrow‐derived primary hMSCs (CD105 + CD166 + CD29 + CD44 + CD14 − CD34 − CD45−) were cultured in osteogenic differentiation medium and expression of OPG protein was evaluated (Figure a,b).…”

“…The combination of these conclusions suggests differing models in the interactions between Col‐GAG and MC‐GAG with osteoprogenitors and osteoclast progenitors (Figures and ). With respect to osteoprogenitors, we previously reported that the necessary mechanism for osteogenic differentiation was an autogenous activation of the canonical BMPR signalling pathway through elevated Smad1/5 phosphorylation for both Col‐GAG and MC‐GAG (Figure , Mechanism 1; Figure , Mechanism 1; Ren, Wei, et al, ; Ren, Tu, et al, ; Zhou et al, ). Whereas Col‐GAG also required activation of the ERK1/2 pathway, MC‐GAG was independent of ERK1/2 for mineralization.…”

“…We have previously reported that a nanoparticulate mineralized collagen glycosaminoglycan (MC‐GAG) material induces efficient osteogenic differentiation of primary human mesenchymal stem cells (hMSCs) and in vivo calvarial healing in a rabbit model in an exogenous growth factor‐independent manner (Ren, Bischoff, et al, ; Ren, Tu, et al, ). We also detailed that an autogenous activation of the canonical bone morphogenetic protein receptor (BMPR) signalling pathway was necessary for osteogenesis of hMSCs on MC‐GAG, whereas the mitogen‐activated protein kinase pathways were not required (Zhou et al, ). Our current work aims to characterize the effects of MC‐GAG on osteoclastogenesis and osteoclast activity.…”

Stiffness of Nanoparticulate Mineralized Collagen Scaffolds Triggers Osteogenesis via Mechanotransduction and Canonical Wnt Signaling

“…Within the realm of calvarial regeneration, we have previously described the ability of an ECM-inspired nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) material to induce osteogenic differentiation of primary, bone marrowderived hMSCs in the absence of exogenous growth factors via autogenously activating the canonical bone morphogenetic protein receptor (BMPR) signaling pathway via phosphorylation of small mothers against decapentaplegic-1/5 (Smad1/5) (12)(13)(14)(15)(16)(17)(18). More importantly, MC-GAG also induced calvarial regeneration as a progenitor cell-free, exogenous growth factor-free material in rabbits in a manner that approached 60% of the stiffness and strength of the outer cortex of native calvarium (15).…”