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Multiomics analysis of cultured mouse periodontal ligament cell-derived extracellular matrix.

ABSTRACT: A comprehensive understanding of the extracellular matrix (ECM) is essential for developing biomimetic ECM scaffolds for tissue regeneration. As the periodontal ligament cell (PDLC)-derived ECM has shown potential for periodontal tissue regeneration, it is vital to gain a deeper understanding of its comprehensive profile. Although the PDLC-derived ECM exhibits extracellular environment similar to that of periodontal ligament (PDL) tissue, details of its molecular composition are lacking. Thus, using a multiomics approach, we systematically analyzed cultured mouse PDLC-derived ECM and compared it to mouse PDL tissue as a reference. Proteomic analysis revealed that, compared to PDL tissue, the cultured PDLC-derived ECM had a lower proportion of fibrillar collagens with increased levels of glycoprotein, corresponding to an immature ECM status. The gene expression signature was maintained in cultured PDLCs and was similar to that in cells from PDL tissues, with additional characteristics representative of naturally occurring progenitor cells. A combination of proteomic and transcriptomic analyses revealed that the cultured mouse PDLC-derived ECM has multiple advantages in tissue regeneration, providing an extracellular environment that closely mimics the environment in the native PDL tissue. These findings provide valuable insights for understanding PDLC-derived ECM and should contribute to the development of biomimetic ECM scaffolds for reliable periodontal tissue regeneration.

SUBMITTER: Kaku M

PROVIDER: S-EPMC10764881 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Multiomics analysis of cultured mouse periodontal ligament cell-derived extracellular matrix.

Kaku Masaru M Thant Lay L Dobashi Azusa A Ono Yoshiki Y Kitami Megumi M Mizukoshi Masaru M Arai Moe M Iwama Hajime H Kitami Kohei K Kakihara Yoshito Y Matsumoto Masaki M Saito Isao I Uoshima Katsumi K

Scientific reports 20240103 1

A comprehensive understanding of the extracellular matrix (ECM) is essential for developing biomimetic ECM scaffolds for tissue regeneration. As the periodontal ligament cell (PDLC)-derived ECM has shown potential for periodontal tissue regeneration, it is vital to gain a deeper understanding of its comprehensive profile. Although the PDLC-derived ECM exhibits extracellular environment similar to that of periodontal ligament (PDL) tissue, details of its molecular composition are lacking. Thus, u ...[more]

PMID: 38172274

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Project description:The periodontal ligament (PDL) is a specialized connective tissue that provides structural support to the tooth and is crucial for oral functions. The mechanical properties of the PDL are mainly derived from the tissue-specific composition and structural characteristics of the extracellular matrix (ECM). The ECM also plays key roles in determining cell fate in the cellular microenvironment thus crucial in the PDL tissue homeostasis. In the present study, we determined the comprehensive ECM profile of mouse molar PDL using laser microdissection and mass spectrometry-based proteomic analysis with ECM-oriented data curation. Additionally, we evaluated changes in the ECM proteome under mechanical loading using a mouse orthodontic tooth movement (OTM) model and analyzed potential regulatory networks using a bioinformatics approach. Proteomic changes were evaluated in reference to the novel second harmonic generation (SHG)-based fiber characterization. Our ECM-oriented proteomics approach succeeded in illustrating the comprehensive ECM profile of the mouse molar PDL. We revealed the presence of type II collagen in PDL, possibly associated with the load-bearing function upon occlusal force. Mechanical loading induced unique architectural changes in collagen fibers along with dynamic compositional changes in the matrisome profile, particularly involving ECM glycoproteins and matrisome-associated proteins. We identified several unique matrisome proteins which responded to the different modes of mechanical loading in PDL. Notably, the proportion of type VI collagen significantly increased at the mesial side, contributing to collagen fibrogenesis. On the other hand, type XII collagen increased at the PDL-cementum boundary of the distal side. Furthermore, a multifaceted bioinformatics approach illustrated the potential molecular cues, including PDGF signaling, that maintain ECM homeostasis under mechanical loading. Our findings provide fundamental insights into the molecular network underlying ECM homeostasis in PDL, which is vital for clinical diagnosis and development of biomimetic tissue-regeneration strategies.

Project description:The major challenge for dentistry is to provide the patient an oral rehabilitation to maintain healthy bone conditions in order to reduce the time for loading protocols. Advancement in implant surface design is necessary to favour and promote the osseointegration process. The surface features of titanium dental implant can promote a relevant influence on the morphology and differentiation ability of mesenchymal stem cells, induction of the osteoblastic genes expression and the release of extracellular matrix (ECM) components. The present study aimed at evaluating the in vitro effects of two different dental implants with titanium surfaces, TEST and CTRL, to culture the human periodontal ligament stem cells (hPDLSCs). Expression of ECM components such as Vimentin, Fibronectin, N-cadherin, Laminin, Focal Adhesion Kinase (FAK) and Integrin beta-1 (ITGB1), and the osteogenic related markers, as runt related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP), were investigated. Human PDLSCs cultured on the TEST implant surface demonstrated a better cell adhesion capability as observed by Scanning Electron Microscopy (SEM) and immunofluorescence analysis. Moreover, immunofluorescence and Western blot experiments showed an over expression of Fibronectin, Laminin, N-cadherin and RUNX2 in hPDLSCs seeded on TEST implant surface. The gene expression study by RT-PCR validated the results obtained in protein assays and exhibited the expression of RUNX2, ALP, Vimentin (VIM), Fibronectin (FN1), N-cadherin (CDH2), Laminin (LAMB1), FAK and ITGB1 in hPDLSCs seeded on TEST surface compared to the CTRL dental implant surface. Understanding the mechanisms of ECM components release and its regulation are essential for developing novel strategies in tissue engineering and regenerative medicine. Our results demonstrated that the impact of treated surfaces of titanium dental implants might increase and accelerate the ECM apposition and provide the starting point to initiate the osseointegration process.

Project description:Rebuilding the tooth-supporting tissues (periodontium) destroyed by periodontitis remains a clinical challenge. Periodontal ligament cells (PDLCs), multipotent cells within the periodontal ligament (PDL), differentiate and form new PDL and mineralized tissues (cementum and bone) during native tissue repair in response to specific extracellular matrix (ECM) cues. Thus, harnessing ECM cues to control PDLC activity ex vivo, and ultimately, to design a PDLC delivery vehicle for tissue regeneration is an important goal. In this study, poly(ethylene glycol) hydrogels were used as a synthetic PDL ECM to interrogate the roles of cell-matrix interactions and cell-mediated matrix remodeling in controlling PDLC activity. Results showed that PDLCs within matrix metalloproteinase (MMP)-degradable hydrogels expressed key PDL matrix genes and showed a six to eightfold increase in alkaline phosphatase (ALP) activity compared with PDLCs in nondegradable hydrogel controls. The increase in ALP activity, commonly considered an early marker of cementogenic/osteogenic differentiation, occurred independent of the presentation of the cell-binding ligand RGD or soluble media cues and remained elevated when inhibiting PDLC-matrix binding and intracellular tension. ALP activity was further increased in softer hydrogels regardless of degradability and was accompanied by an increase in PDLC volume. However, scaffolds that fostered PDLC ALP activity did not necessarily promote hydrogel ECM mineralization. Rather, matrix mineralization was greatest in stiffer, MMP-degradable hydrogels and required the presence of soluble media cues. These divergent outcomes illustrate the complexity of the PDLC response to ECM cues and the limitations of current scaffold materials. Nevertheless, key biomaterial design principles for controlling PDLC activity were identified for incorporation into scaffolds for periodontal tissue regeneration. Impact statement Engineered scaffolds are an attractive approach for delivering periodontal ligament cells (PDLCs) to rebuild the tooth-supporting tissues. Replicating key extracellular matrix (ECM) cues within tissue engineered scaffolds may maximize PDLC potential. However, the identity of important ECM cues and how they can be harnessed to control PDLC activity is still unknown. In this study, matrix degradability, cell-matrix binding, and stiffness were varied using synthetic poly(ethylene glycol) hydrogels for three-dimensional PDLC culture. PDLCs exhibited dramatic and divergent responses to these cues, supporting further investigation of ECM-replicating scaffolds for control of PDLC behavior and periodontal tissue regeneration.

Project description:ObjectivePer-implantitis is one of the implant treatment complications. Dentists have failed to restore damaged periodontium by using conventional therapies. Tissue engineering (stem cells, scaffold and growth factors) aims to reconstruct natural tissues. The paper aimed to isolate both periodontal ligament stem cells (PDLSCs) and bone marrow mesenchymal stem cells (BMMSCs) and use them in a co-culture method to create three-layered cell sheets for reconstructing natural periodontal ligament (PDL) tissue.Materials and methodsBMMSCs were isolated from rabbit tibia and femur, and PDLSC culture was established from the lower right incisor. The cells were co-cultured to induce BMMSC differentiation into PDL cells. Cell morphology, stem cells and PDL-specific markers (CD90, CD34, and periostin) were also detected using immunofluorescent assay. Co-cultured cell monolayers were detached using temperature-responsive tissue culture dishes and collagen graft to create the three-layer construct. The 3D-engineered tissue was examined histologically and by field emission scanning electron microscopy (FESEM).ResultsBMMSCs co-cultured with PDLSCs successfully induced more PDL cells. The newly induced PDL cells exhibited periostin and CD90 expression. Fluorescence green intensity was measured for the co-cultured cells that were stained with periostin, the mean fluorescence green intensity (periostin expression) was significantly higher for the newly induced PDL cells after 1, 2, and 3 weeks when compared with control (BM-MSCs), at 21 days non-significant difference was measured when compared with control (PDLSCs).The results showed the successful formation of 3D multilayer PDL tissue. Histological cross-section showed cell sheets and the stable adhesion between them. FESEM examination was conducted for the cross-section, showing three-layered cell sheets with stable adhesion between cells.ConclusionsThe results of this paper report that the three layered-cell sheets were successfully constructed by the novel use of collagen graft as a scaffold to be used in treatment of periodontitis and to envelop the dental implants to create biohybrid implant.

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Multiomics analysis of cultured mouse periodontal ligament cell-derived extracellular matrix.

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Multiomics analysis of cultured mouse periodontal ligament cell-derived extracellular matrix.

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