Abstract
Dental pulp stem cells (DPSCs) are increasingly being advocated for regenerative medicine-based therapies. However, significant heterogeneity in the genotypic/phenotypic properties of DPSC subpopulations exist, influencing their therapeutic potentials. As most studies have established DPSC heterogeneity using 2D culture approaches, we investigated whether heterogeneous DPSC proliferative and contraction/remodelling capabilities were further evident within 3D type I collagen gels in vitro. DPSC subpopulations were isolated from human third molars and identified as high/low proliferative and multipotent/unipotent, following in vitro culture expansion and population doubling (PD) analysis. High proliferative/multipotent DPSCs, such as A3 (30 PDs and 80 PDs), and low proliferative/unipotent DPSCs, such as A1 (17 PDs), were cultured in collagen gels for 12 days, either attached or detached from the surrounding culture plastic. Collagen architecture and high proliferative/multipotent DPSC morphologies were visualised by Scanning Electron Microscopy and FITC-phalloidin/Fluorescence Microscopy. DPSC proliferation (cell counts), contraction (% diameter reductions), and remodelling (MMP-2/MMP-9 gelatin zymography) of collagen gels were also evaluated. Unexpectedly, no proliferation differences existed between DPSCs, A3 (30 PDs) and A1 (17 PDs), although A3 (80 PDs) responses were significantly reduced. Despite rapid detached collagen gel contraction with A3 (30 PDs), similar contraction rates were determined with A1 (17 PDs), although A3 (80 PDs) contraction was significantly impaired. Gel contraction correlated to distinct gelatinase profiles. A3 (30 PDs) possessed superior MMP-9 and comparable MMP-2 activities to A1 (17 PDs), whereas A3 (80 PDs) had significantly reduced MMP-2/MMP-9. High proliferative/multipotent DPSCs, A3 (30 PDs), further exhibited fibroblast-like morphologies becoming polygonal within attached gels, whilst losing cytoskeletal organization and fibroblastic morphologies in detached gels. This study demonstrates that heterogeneity exists in the gel contraction and MMP expression/activity capabilities of DPSCs, potentially reflecting differences in their abilities to degrade biomaterial scaffolds and regulate cellular functions in 3D environments and their regenerative properties overall. Thus, such findings enhance our understanding of the molecular and phenotypic characteristics associated with high proliferative/multipotent DPSCs.
Highlights
Adult human dental pulp stem cells (DPSCs) are increasingly being characterised and evaluated as a viable mesenchymal stem cell (MSC) source in the development of effective regenerative medicine-based therapies for clinical use [1,2,3]
This study aimed to determine whether the well-established variations in the proliferative, differentiation and other stem cell properties between different DPSC subpopulations in the 2D monolayer culture were further identifiable in 3D cultures within type I collagen gels
As with previous studies highlighting the phenotypic and genotypic heterogeneity which exists between high proliferative/multipotent and low proliferative/unipotent DPSC subpopulations utilising 2D monolayer cultures, this study demonstrates that heterogeneity is evident in terms of the gel contraction capabilities and matrix metalloproteinases (MMPs) expression/activity profiles of individual DPSC subpopulations, following seeding within 3D type I collagen gels
Summary
Adult human dental pulp stem cells (DPSCs) are increasingly being characterised and evaluated as a viable mesenchymal stem cell (MSC) source in the development of effective regenerative medicine-based therapies for clinical use [1,2,3]. Such conclusions are based on DPSCs being readily accessible from the permanent dentition, in addition to their selfrenewal, clonogenicity, and multilineage (e.g., osteogenic, chondrogenic, adipogenic, myogenic, and neurogenic) differentiation capabilities, comparable to those established for bone marrow-derived MSCs [4,5,6]. Most natural and semisynthetic biomaterial scaffolds are designed to undergo gradual remodelling and degradation by matrix metalloproteinases (MMPs), such as gelatinases MMP-2 and MMP-9, to be replaced by the newly synthesized tissue whilst still providing a functional role in supporting MSC activity [13,14,15,16]
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