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Hyaluronan-Based Gel Promotes Human Dental Pulp Stem Cells Bone Differentiation by Activating YAP/TAZ Pathway.

ABSTRACT: Hyaluronans exist in different forms, accordingly with molecular weight and degree of crosslinking. Here, we tested the capability to induce osteogenic differentiation in hDPSCs (human dental pulp stem cells) of three hyaluronans forms: linear pharmaceutical-grade hyaluronans at high and (HHA) low molecular weight (LHA) and hybrid cooperative complexes (HCC), containing both sizes. hDPSCs were treated with HHA, LHA, HCC for 7, 14 and 21 days. The effects of hyaluronans on osteogenic differentiation were evaluated by qRT-PCR and WB of osteogenic markers and by Alizarin Red S staining. To identify the involved pathway, CD44 was analyzed by immunofluorescence, and YAP/TAZ expression was measured by qRT-PCR. Moreover, YAP/TAZ inhibitor-1 was used, and the loss of function of YAP/TAZ was evaluated by qRT-PCR, WB and immunofluorescence. We showed that all hyaluronans improves osteogenesis. Among these, HCC is the main inducer of osteogenesis, along with overexpression of bone related markers and upregulating CD44. We also found that this biological process is subordinate to the activation of YAP/TAZ pathway. We found that HA's molecular weight can have a relevant impact on HA performance for bone regeneration, and we unveil a new molecular mechanism by which HA acts on stem cells.

SUBMITTER: La Noce M

PROVIDER: S-EPMC8616223 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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Hyaluronan-Based Gel Promotes Human Dental Pulp Stem Cells Bone Differentiation by Activating YAP/TAZ Pathway.

La Noce Marcella M Stellavato Antonietta A Vassallo Valentina V Cammarota Marcella M Laino Luigi L Desiderio Vincenzo V Del Vecchio Vitale V Nicoletti Giovanni Francesco GF Tirino Virginia V Papaccio Gianpaolo G Schiraldi Chiara C Ferraro Giuseppe Andrea GA

Cells 20211026 11

<h4>Background</h4>Hyaluronans exist in different forms, accordingly with molecular weight and degree of crosslinking. Here, we tested the capability to induce osteogenic differentiation in hDPSCs (human dental pulp stem cells) of three hyaluronans forms: linear pharmaceutical-grade hyaluronans at high and (HHA) low molecular weight (LHA) and hybrid cooperative complexes (HCC), containing both sizes.<h4>Methods</h4>hDPSCs were treated with HHA, LHA, HCC for 7, 14 and 21 days. The effects of hyal ...[more]

PMID: 34831122

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Project description:BackgroundRadial extracorporeal shockwave (r-ESW), an innovative and noninvasive technique, is gaining increasing attention in regenerative medicine due to its mechanobiological effects. Subchondral bone stem/progenitor cells (SCB-SPCs), originating from the pivotal zone of the osteochondral unit, have been shown to have multipotency and self-renewal properties. However, thus far, little information is available regarding the influences of r-ESW on the biological properties of SCB-SPCs and their therapeutic effects in tissue regeneration.MethodsSCB-SPCs were isolated from human knee plateau osteochondral specimens and treated with gradient doses of r-ESW in a suspension stimulation system. The optimized parameters for SCB-SPC self-renewal were screened out by colony-forming unit fibroblast assay (CFU-F). Then, the effects of r-ESW on the proliferation, apoptosis, and multipotency of SCB-SPCs were evaluated. Moreover, the repair efficiency of radial shockwave-preconditioned SCB-SPCs was evaluated in vivo via an osteochondral defect model. Potential mechanisms were explored by western blotting, confocal laser scanning, and high-throughput sequencing.ResultsThe CFU-F data indicate that r-ESW could augment the self-renewal of SCB-SPCs in a dose-dependent manner. The CCK-8 and flow cytometry results showed that the optimized shockwave markedly promoted SCB-SPC proliferation but had no significant influence on cell apoptosis. Radial shockwave exerted no significant influence on osteogenic capacity but strongly suppressed adipogenic ability in the current study. For chondrogenic potentiality, the treated SCB-SPCs were mildly enhanced, while the change was not significant. Importantly, the macroscopic scores and further histological analysis strongly demonstrated that the in vivo therapeutic effects of SCB-SPCs were markedly improved post r-ESW treatment. Further analysis showed that the cartilage-related markers collagen II and proteoglycan were expressed at higher levels compared to their counterpart group. Mechanistic studies suggested that r-ESW treatment strongly increased the expression of YAP and promoted YAP nuclear translocation in SCB-SPCs. More importantly, self-renewal was partially blocked by the YAP-specific inhibitor verteporfin. Moreover, the high-throughput sequencing data indicated that other self-renewal-associated pathways may also be involved in this process.ConclusionWe found that r-ESW is capable of promoting the self-renewal of SCB-SPCs in vitro by targeting YAP activity and strengthening its repair efficiency in vivo, indicating promising application prospects.

Project description:The lack of vasculogenesis often hampers the survivability and integration of newly engineered tissue grafts within the host. Autologous endothelial cells (ECs) are an ideal cell source for neovascularization, but they are limited by their scarcity, lack of proliferative capacity, and donor site morbidity upon isolation. The objective of this study was to determine whether differentiation of human dental pulp stem cells (DPSCs) into the endothelial lineage can be enhanced by recombinant ETV2 overexpression. DPSCs were extracted from fresh dental pulp tissues. ETV2 overexpression in DPSCs was achieved by lentiviral infection and cellular morphological changes were evaluated. The mRNA and protein expression levels of endothelial-specific markers were assessed through quantitative real-time polymerase chain reaction, western blot, immunofluorescence staining, and flow cytometry. The tube formation assay and Matrigel plug assay were also performed to evaluate the angiogenic potential of the ETV2-transduced cells in vitro and in vivo, respectively. Additionally, proteomic analysis was performed to analyze global changes in protein expression following ETV2 overexpression. After lentiviral infection, ETV2-overexpressing DPSCs showed endothelial-like morphology. Compared with control DPSCs, significantly higher mRNA and protein expression levels of endothelial-specific genes, including CD31, VE-Cadherin, VEGFR1, and VEGFR2, were detected in ETV2-overexpressing DPSCs. Moreover, ETV2 overexpression enhanced capillary-like tube formation on Matrigel in vitro, as well as neovascularization in vivo. In addition, comparative proteomic profiling showed that ETV2 overexpression upregulated the expression of vascular endothelial growth factor (VEGF) receptors, which was indicative of increased VEGF signaling. Taken together, our results indicate that ETV2 overexpression significantly enhanced the endothelial differentiation of DPSCs. Thus, this study shows that DPSCs can be a promising candidate cell source for tissue engineering applications.

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Hyaluronan-Based Gel Promotes Human Dental Pulp Stem Cells Bone Differentiation by Activating YAP/TAZ Pathway.

Publications

Hyaluronan-Based Gel Promotes Human Dental Pulp Stem Cells Bone Differentiation by Activating YAP/TAZ Pathway.

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