European cells & materials最新文献

The objective was to compare different dental splint models and materials for inducing abnormal loading on the gross morphology and histological appearance of the mandibular condylar processes of Sprague Dawley rats. Three different types of dental splints (resin molar, aluminum incisor, stainless-steel incisor) were placed unilaterally to induce occlusal perturbation for 4 weeks. At that time, mandibular condylar processes were assessed by gross appearance and histology. Quantitative measurements were also conducted on the hematoxylin and eosin images for condyle shape. The results showed that although the condylar cartilage was affected by all splint types, the resin molar splint was associated with the most extensive mandibular condylar process remodeling, which was primarily a slant (skewness) of the lateral aspect of the condylar process. Additionally, quantitative measurements on the histological specimens demonstrated that the split and tilt angle of the left (ipsilateral) condylar processes in the resin molar group (124.8 ± 12.7° and 104.1 ± 12.7°, respectively) increased significantly (p < 0.05) when compared to right (contralateral) condylar processes (104.7 ± 5.8°and 91.6 ± 4.4°, respectively). However, no changes were noted on the thickness of the fibrocartilage layer at medial, central, and lateral regions of the condylar process. Another major finding is the high variability of morphology of the naïve animals. Future studies will assess the impact of longer durations of splinting, age, and sex on the remodeling of the mandibular condylar process, allowing for the development of diagnostics and therapies.

The pathogenesis of posterior longitudinal ligament ossification (OPLL) remains inadequately understood. Mechanical stimulation is one of the important pathogenic factors in OPLL. As one of the mechanical stimulation transduction signals, the yes-associated protein (YAP) interacts with the Wnt/β-catenin signalling pathway, which plays an important role in osteogenic differentiation. This study aimed to demonstrate the role of YAP-Wnt/β-catenin axis in cell differentiation induced by mechanical stress. Primary cells extracted from posterior longitudinal ligament tissues from OPLL or non-OPLL patients were subjected to sinusoidal uniaxial cyclic stretch (5 %, 0.5 Hz, 3 d). The expression of runt-related transcription factor 2, collagen I, osterix, osteocalcin and alkaline phosphatase were compared between the static and the experimental groups. In addition, the cytoskeleton was detected using phalloidin staining while YAP phosphorylation states and nuclear location were identified using immunofluorescence. The results showed that mechanical stretching loading increased the expression of osteogenic genes and proteins in the OPLL group, while it had no significant effect on the control group. When OPLL cells were stretched, YAP exhibited an obvious nuclear translocation and the Wnt/β-catenin pathway was activated. Knocking down YAP or β-catenin could weaken the impact upon osteogenic differentiation induced by mechanical stimulation. YAP-mediated mechanical stimulation promoted osteogenic differentiation of OPLL cells through Wnt/β-catenin pathway and this progress was independent of the Hippo pathway.

Periodontitis is a progressive disease that ultimately leads to bone and tooth loss. A major consequence of periodontal disease is the inability to regain lost bone in the periodontium. The importance was demonstrated of glucose-regulated protein-78 (GRP78) in the osteogenic differentiation of periodontal ligament stem cells and their potential use for regeneration of the periodontium. Previous studies have shown the relationship between GRP78 and dentine matrix protein-1 (DMP1). The importance of this receptor-ligand complex in supporting the process of osteogenesis and angiogenesis was confirmed in this study. To show the function of GRP78 in mineralised tissues, transgenic periodontal ligament stem cells (PDLSCs) were generated in which GRP78 was either overexpressed or silenced. Gene expression analysis of the cells cultured under osteogenic conditions showed an increase in key osteogenic genes with the overexpression of GRP78. RNA-Seq analysis was also performed to understand the transcriptome profile associated with genotype changes. Using the database for annotation, visualisation, and integration discovery (DAVID) for the functional enrichment analysis of differentially expressed genes, the upregulation of genes promoting osteogenesis and angiogenesis with GRP78 overexpression was demonstrated. Alizarin red staining and scanning electron microscopy analysis revealed matrix mineralisation with increased calcium deposition in GRP78 overexpressing cells. The in vivo osteogenic and angiogenic function of GRP78 was shown using a subcutaneous implantation rodent model. The results suggested that GRP78 in PDLSCs can regulate the expression of both osteogenesis and angiogenesis. Therefore, GRP78 could be considered as a therapeutic target for repair of diseased periodontium.

A critical component of the temporomandibular joint (TMJ) is the fibrocartilage articular disc (AD). Researchers have attempted to regenerate the AD to alleviate TMJ osteoarthritis but alternative cell sources for use in AD regenerative approaches are needed due to insufficient extracellular matrix (ECM) production by total articular disc cells (TACs). Tissue-specific progenitor cells have been identified in many tissues. The aim of the present study was to identify adult multipotent progenitor cells within the AD suitable for regenerative medicine applications. A novel AD progenitor cell population was identified in rhesus macaques. Clonally derived articular disc progenitor cells (ADPs) were isolated using fibronectin differential cell adhesion. ADPs represent between 1 and 3 % of the TAC population and are capable of in vitro expansion beyond 60 population doublings. ADPs were characterized using osteogenic, adipogenic, and fibrochondrogenesis differentiation assays. Clones exhibited phenotypic plasticity, differentiating into osteocytes, adipocytes, and fibrochondrocytes. ECM secretion profiles following fibrochondrogenic differentiation were assessed using immunohistochemistry (IHC), fluorescently activated cell sorting (FACS), total collagen, and glycosaminoglycan (GAG) assays and compared with TACs, articular cartilage progenitor cells (ACPs), tendon progenitor cells (TPCs) and bone-marrow-derived mesenchymal stem cells (BMMSCs). ADP pellet cultures produced a biochemical phenotype similar to native AD tissue, with production of versican (VCAN) and collagen types I, II, III, and VI (COL1, COL2, COL3, COL6). However, clonally derived ADP cell lines produced different amounts of ECM and exhibited different expansion potentials. These findings indicated flexibility in clone selection for potential regenerative strategies to recapitulate native anisotropy.

Skeletal muscle contractions are critical for normal skeletal growth and morphogenesis but it is unclear how the detrimental effects of absent muscle on the bones and joints change over time. Joint shape and cavitation as well as rudiment length and mineralisation were assessed in multiple rudiments at two developmental stages [Theiler stage (TS)24 and TS27] in the splotch-delayed "muscle-less limb" mouse model and littermate controls. Chondrocyte morphology was quantified in 3D in the distal humerus at the same stages. As development progressed, the effects of absent muscle on all parameters except for cavitation become less severe. All major joints in muscle-less limbs were abnormally shaped at TS24, while, by TS27, most muscle-less limb joint shapes were normal or nearly normal. In contrast, any joints that were fused at TS24 did not cavitate by TS27. At TS24, chondrocytes in the distal humerus were significantly smaller in the muscle-less limbs than in controls, while by TS27, chondrocyte volume was similar between the two groups, offering a cell-level mechanism for the partial recovery in shape of muscle-less limbs. Mineralisation showed the most pronounced changes over gestation. At TS24, all muscle-less rudiments studied had less mineralisation than the controls, while at TS27, muscle-less limb rudiments had mineralisation extents equivalent to controls. In conclusion, the effects of muscle absence on prenatal murine skeletogenesis reduced in severity over gestation. Understanding how mammalian bones and joints continue to develop in an environment with abnormal fetal movements provides insights into conditions including hip dysplasia and arthrogryposis.

Extensive extracellular matrix production and increased cell-matrix adhesion by bone marrow stromal cells (BMSCs) are hallmarks of fibrotic alterations in the vertebral bone marrow known as Modic type 1 changes (MC1). MC1 are associated with non-specific chronic low-back pain. To identify treatment targets for MC1, in vitro studies using patient BMSCs are important to reveal pathological mechanisms. For the culture of BMSCs, fibroblast growth factor 2 (FGF2) is widely used. However, FGF2 has been shown to suppress matrix synthesis in various stromal cell populations. The aim of the present study was to investigate whether FGF2 affected the in vitro study of the fibrotic pathomechanisms of MC1-derived BMSCs. Transcriptomic changes and changes in cell-matrix adhesion of MC1-derived BMSCs were compared to intra-patient control BMSCs in response to FGF2. RNA sequencing and quantitative real-time polymerase chain reaction revealed that pro-fibrotic genes and pathways were not detectable in MC1-derived BMSCs when cultured in the presence of FGF2. In addition, significantly increased cell-matrix adhesion of MC1-derived BMSCs was abolished in the presence of FGF2. In conclusion, the data demonstrated that FGF2 overrides key pro-fibrotic features of MC1 BMSCs in vitro. Usage of FGF2-supplemented media in studies of fibrotic mechanisms should be critically evaluated as it could override normally dominant biological and biophysical cues.

Chronic tendinopathy represents a growing healthcare burden in the ageing global population. Curative therapies remain elusive as the mechanisms that underlie chronic inflammation in tendon disease remain unclear. Identifying and isolating key pathogenic and reparative cells is essential in developing precision therapies and implantable materials for improved tendon healing. Multiple discrete human tendon cell populations have been previously described ex vivo. To determine if these populations persist in vitro, healthy human hamstring tenocytes were cultured for 8 d on either tissue culture plastic or aligned electrospun fibres of absorbable polydioxanone. Novel single-cell surface proteomics combined with unbiased single-cell transcriptomics (CITE-Seq) was used to identify discrete tenocyte populations. 6 cell populations were found, 4 of which shared key gene expression determinants with ex vivo human cell clusters: PTX3_PAPPA, POSTN_SCX, DCN_LUM and ITGA7_NES. Surface proteomics found that PTX3_PAPPA cells were CD10+CD26+CD54+. ITGA7_NES cells were CD146+ and POSTN_SCX cells were CD90+CD95+CD10+. Culture on the aligned electrospun fibres favoured 3 cell subtypes (DCN_LUM, POSTN_SCX and PTX3_ PAPPA), promoting high expression of tendon-matrix-associated genes and upregulating gene sets enriched for TNF-a and IL-6/STAT3 signalling. Discrete human tendon cell subpopulations persisted in in vitro culture and could be recognised by specific gene and surface-protein signatures. Aligned polydioxanone fibres promoted the survival of 3 clusters, including pro-inflammatory PTX3-expressing CD10+CD26+CD54+ cells found in chronic tendon disease. These results improved the understanding of preferred culture conditions for different tenocyte subpopulations and informed the development of in vitro models of tendon disease.