European cells & materials最新文献

This study presents a simple and cost-effective model using microparticles to simulate the bacterial distribution pattern in soft tissue after low- and high-pressure irrigation. Silica coated iron microparticles [comparable diameter (1 µm) and weight (0.8333 pg) to Staphylococcus aureus] were applied to the surface of twenty fresh human muscle tissue samples in two amputated lower legs. Particle dissemination into deep tissue layers as an undesired side effect was investigated in four measuring fields as positive control (PC) as well as after performing pulsatile high-pressure (HP, 8 measuring fields) and low-pressure flushing (LP, 8 measuring fields). Five biopsies were taken out of each measuring field to get a total number of 100 biopsies. After histological and digital image processing, the specimens were analysed, and all incomplete sections were excluded. A special detection algorithm was parameterised using the open source bioimage analysis software QuPath. The application of this detection algorithm enabled automated counting and detection of the particles with a sensitivity of 95 % compared to manual counts. Statistical analysis revealed significant differences (p < 0.05) in our three different sample groups: HP (M = 1608, S = 302), LP (M = 2176, SD = 609) and PC (M = 4011, SD = 686). While both HP and LP flushing techniques are able to reduce the number of bacteria, a higher effectiveness is shown for HP irrigation. Nevertheless, a challenge for the validity of the study is the use of dead tissue and therefore a possible negative influence of high-pressure irrigation on tissue healing and further dispersion of particles cannot be evaluated.

While it is known that the degenerated intervertebral disc (IVD) is one of the primary reasons for low-back pain and subsequent need for medical care, there are currently no established effective methods for direct treatment. Nuclear factor-κB (NF-κB) is a transcription factor that regulates various genes' expression, among which are inflammatory cytokines, in many tissues including the IVD. NF-κB decoy is an oligodeoxynucleotide containing the NF-κB binding site that entraps NF-κB subunits, resulting in suppression of NF-κB activity. In the present preclinical study, NF-κB decoy was injected into degenerated IVDs using the rabbit anular-puncture model. In terms of distribution, NF-κB decoy persisted in the IVDs up to at least 4 weeks after injection. The remaining amount of NF-κB decoy indicated that it fit a double-exponential-decay equation. Investigation of puncture-caused degeneration of IVDs showed that NF-κB decoy injection recovered, dose-dependently, the reduced disc height that was associated with reparative cell cloning and morphological changes, as assessed through histology. Gene expression, by quantitative real-time polymerase chain reaction (qRT-PCR), showed that NF-κB decoy attenuated inflammatory gene expression, such as that of interleukin-1 and tumor necrosis factor-α, in rabbit degenerated IVDs. NF-κB decoy also reduced the pain response as seen using the "pain sensor" nude rat xenograft-radiculopathy model. This is the first report demonstrating that NF-κB decoy suppresses the inflammatory response in degenerated IVDs and restores IVD disc height loss. Therefore, the intradiscal injection of NF-κB decoy may have the potential as an effective therapeutic strategy for discogenic pain associated with degenerated IVDs.

Insulin-like growth factor I (IGF-I) is essential for muscle and bone development and a primary mediator of growth hormone (GH) actions. While studies have elucidated the importance of IGF-I specifically in muscle or bone development, few studies to date have evaluated the relationship between muscle and bone modulated by IGF-I in vivo, during post-natal growth. Mice with muscle-specific IGF-I overexpression (mIgf1+/+) were utilised to determine IGF-I- and muscle-mass-dependent effects on craniofacial skeleton development during post-natal growth. mIgf1+/+ mice displayed accelerated craniofacial bone growth when compared to wild-type animals. Virus-mediated expression of IGF-I targeting the masseter was performed to determine if post-natal modulation of IGF-I altered mandibular structures. Increased IGF-I in the masseter affected the mandibular base plane angle in a lateral manner, increasing the width of the mandible. At the cellular level, increased muscle IGF-I also accelerated cartilage thickness in the mandibular condyle. Importantly, mandibular length changes associated with increased IGF-I were not present in mice with genetic inhibition of muscle IGF-I receptor activity. These results demonstrated that muscle IGF-I could indirectly affect craniofacial growth through IGF-I-dependent increases in muscle hypertrophy. These findings have clinical implications when considering IGF-I as a therapeutic strategy for craniofacial disorders.

Dentineogenesis starts on odontoblasts, which synthesise and secrete non-collagenous proteins (NCPs) and collagen. When dentine is injured, dental pulp progenitors/mesenchymal stem cells (MSCs) can migrate to the injured area, differentiate into odontoblasts and facilitate formation of reactionary dentine. Dental pulp progenitor cell/MSC differentiation is controlled at given niches. Among dental NCPs, dentine sialophosphoprotein (DSPP) is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family, whose members share common biochemical characteristics such as an Arg-Gly-Asp (RGD) motif. DSPP expression is cell- and tissue-specific and highly seen in odontoblasts and dentine. DSPP mutations cause hereditary dentine diseases. DSPP is catalysed into dentine glycoprotein (DGP)/sialoprotein (DSP) and phosphoprotein (DPP) by proteolysis. DSP is further processed towards active molecules. DPP contains an RGD motif and abundant Ser-Asp/Asp-Ser repeat regions. DPP-RGD motif binds to integrin αVβ3 and activates intracellular signalling via mitogen-activated protein kinase (MAPK) and focal adhesion kinase (FAK)-ERK pathways. Unlike other SIBLING proteins, DPP lacks the RGD motif in some species. However, DPP Ser-Asp/Asp-Ser repeat regions bind to calcium-phosphate deposits and promote hydroxyapatite crystal growth and mineralisation via calmodulin-dependent protein kinase II (CaMKII) cascades. DSP lacks the RGD site but contains signal peptides. The tripeptides of the signal domains interact with cargo receptors within the endoplasmic reticulum that facilitate transport of DSPP from the endoplasmic reticulum to the extracellular matrix. Furthermore, the middle- and COOH-terminal regions of DSP bind to cellular membrane receptors, integrin β6 and occludin, inducing cell differentiation. The present review may shed light on DSPP roles during odontogenesis.

Implant-associated infections are the primary cause of complications following orthopaedic surgery. Due to biofilm and persister formation, current treatments, i.e. surgical debridement followed by antibiotics, often fail. There is an urgent need for alternative strategies to combat such infections. Therefore, the present study investigated the effects of non-contact induction heating (NCIH), the antimicrobial peptide SAAP-148 and combinations thereof on bacterial counts in 7 d mature biofilms and in persister-enriched biofilms of methicillin-resistant Staphylococcus aureus (MRSA) on titanium-aluminium-niobium (TAN) discs. Enrichment of persisters was achieved by daily exposure of mature biofilms to high doses of rifampicin and ciprofloxacin for 3 consecutive days. To heat up the TAN discs, a miniaturised induction heater was built and successfully validated. Using this apparatus, NCIH resulting in surface temperatures up to 85 °C eradicated all the bacteria in immature biofilms but not in mature biofilms, whereas persisters were already eliminated at surface temperatures ≥ 70 °C. SAAP-148 at concentrations > 25.6 µmol/L reduced the persister counts in antibiotics-exposed, mature biofilms. As surface temperatures > 60 °C can have detrimental effects on the surrounding tissues, the maximum temperature of NCIH used in combination with SAAP-148 on persisters was set to 60 °C. Results revealed that this combination was slightly more effective than the peptide or NCIH alone in eliminating biofilm-embedded persisters. NCIH and SAAP-148 can be applied both invasively and non-invasively in various treatment scenarios. Together, combinations of NCIH and SAAP-148 might be a promising treatment strategy to combat metal-implant-associated infections.

Periodontitis remains an unsolved oral disease, prevalent worldwide and resulting in tooth loss due to dysfunction of the periodontal ligament (PDL), a tissue connecting the tooth root with the alveolar bone. A scaffold-free three-dimensional (3D) organoid model for in vitro tenogenesis/ligamentogeneis has already been described. As PDL tissue naturally arises from the dental follicle, the aim of this study was to investigate the ligamentogenic differentiation potential of dental follicle cells (DFCs) in vitro by employing this 3D model. Human primary DFCs were compared, in both two- and three-dimensions, to a previously published PDL- hTERT cell line. The 3D organoids were evaluated by haematoxylin and eosin, 4',6-diamidino-2-phenylindole and F-actin staining combined with detailed histomorphometric analyses of cell-row structure, angular deviation and cell density. Furthermore, the expression of 48 tendon/ligament- and multilineage-related genes was evaluated using quantitative polymerase chain reaction, followed by immunofluorescent analyses of collagen 1 and 3. The results showed that both cell types were successful in the formation of scaffold-free 3D organoids. DFC organoids were comparable to PDL-hTERT in terms of cell density; however, DFCs exhibited superior organoid morphology, cell-row organisation (p < 0.0001) and angular deviation (p < 0.0001). Interestingly, in 2 dimensions as well as in 3D, DFCs showed significantly higher levels of several ligament- related genes compared to the PDL-hTERT cell line. In conclusion, DFCs exhibited great potential to form PDL-like 3D organoids in vitro suggesting that this strategy can be further developed for functional PDL engineering.

Mesenchymal stem/stromal cell (MSC)-based therapies have been proposed for back pain and disc degeneration, despite limited knowledge on their mechanism of action. The impact of MSCs/their secretome on annulus fibrosus (AF) cells and tissue was analysed in bovine AF organ cultures (AF-OCs) exposed to upper-physiological cyclic tensile strain (CTS, 9 %, 1 Hz, 3 h/d) and interleukin (IL)-1β in a custom-made device. A 4 d treatment of the CTS + IL-1β-stimulated AF-OCs with MSC secretome downregulated the expression of inflammation markers [IL-6, IL-8, prostaglandin-endoperoxide synthase 2 (PTGS2)], complement system regulators [cluster of differentiation (CD)46, CD55, CD59] and matrix metalloproteinase 1 but also of tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2) and collagen type I. At the protein level, it was confirmed that IL-6, MMP-3 and collagen content was decreased in AF-OCs treated with the MSC secretome compared to the CTS + IL-1β stimulation alone. 9 d after treatment, a biomechanical peel-force test showed that the annular adhesive strength was significantly decreased by the MSC secretome treatment. Overall, MSC secretome had a stronger impact on AF tissue than MSCs in co-culture. The secretome contributed to a decrease in the inflammatory and catabolic status of AF cells activated by CTS + IL-1β and played a role in the regulation of the complement system. However, it also contributed to a decrease in collagen at the gene/protein level and in AF mechanical strength compared to the CTS + IL-1β stimulation alone. Therefore, the use of MSC secretome requires further investigation regarding its influence on disc matrix properties.

In vitro models aim to recapitulate the in vivo situation. To more closely mimic the knee joint environment, current in vitro models need improvements to reflect the complexity of the native tissue. High molecular weight hyaluronan (hMwt HA) is one of the most abundant bioactive macromolecules in healthy synovial fluid, while shear and dynamic compression are two joint-relevant mechanical forces. The present study aimed at investigating the concomitant effect of joint-simulating mechanical loading (JSML) and hMwt HA-supplemented culture medium on the chondrogenic differentiation of primary human bone-marrow-derived mesenchymal stem cells (hBM-MSCs). hBM-MSC chondrogenesis was investigated over 28 d at the gene expression level and total DNA, sulphated glycosaminoglycan, TGF-β1 production and safranin O staining were evaluated. The concomitant effect of hMwt HA culture medium and JSML significantly increased cartilage-like matrix deposition and sulphated glycosaminoglycan synthesis, especially during early chondrogenesis. A stabilisation of the hBM-MSC-derived chondrocyte phenotype was observed through the reduced upregulation of the hypertrophic marker collagen X and an increase in the chondrogenic collagen type II/X ratio. A combination of JSML and hMwt HA medium better reflects the complexity of the in vivo synovial joint environment. Thus, JSML and hMwt HA medium will be two important features for joint-related culture models to more accurately predict the in vivo outcome, therefore reducing the need for animal studies. Reducing in vitro artefacts would enable a more reliable prescreening of potential cartilage repair therapies.

Diamond-like carbon (DLC) is a biocompatible material that has many potential biomedical applications, including in orthopaedics. DLC layers doped with Cr at atomic percent (at.%) of 0, 0.9, 1.8, 7.3, and 7.7 at.% were evaluated with reference to their osteoinductivity with human bone marrow mesenchymal stromal cells (hMSCs), immune activation potential with RAW 264.7 macrophage-like cells, and their effect on apoptosis in Saos-2 human osteoblast-like cells and neonatal human dermal fibroblasts (NHDFs). At mRNA level, hMSCs on DLC doped with 0.9 and 7.7 at.% of Cr reached higher maximum values of both RUNX2 and alkaline phosphatase. An earlier onset of mRNA production of type I collagen and osteocalcin was also observed on these samples; they also supported the production of both type I collagen and osteocalcin. RAW 264.7 macrophages were screened using a RayBio™ Human Cytokine Array for cytokine production. 10 cytokines were at a concentration more than 2 × as high as the concentration of a positive control, but the values for the DLC samples were only moderately higher than the values on glass. NHDF cells, but not Saos-2 cells, had a higher expression of pro-apoptotic markers Bax and Bim and a lower expression of anti-apoptotic factor BCL-XL in proportion to the Cr content. Increased apoptosis was also proven by annexin V staining. These results show that a Cr-doped DLC layer with a lower Cr content can act as an osteoinductive material with relatively low immunogenicity, but that a higher Cr content can induce cell apoptosis.

Ideal restoration material for caries would allow attachment of gingival epithelia. The attachment of epithelial cells to specimens of the 4 most commercially used well- or partially-cured resin composites, with and without TEGDMA, was assessed. Effects of resin composite on the Ca9-22 gingival epithelial cell-line were assessed by measuring the cytotoxicity, viability and gene expression for attachment, apoptosis, ROS-production, pro-inflammatory cytokines, and matrix metalloproteinases. As controls, cells on tissue culture plastic or bovine tooth enamel specimens were used. Significantly less cell attachment was measured on freshly made resin-composite specimens. Concomitantly, significantly higher cytotoxicity was measured in the presence of freshly made resin-composite specimens. However, after 8 d of leakage, the cell attachment to and cytotoxicity of the resin composite was comparable to bovine tooth enamel. Significantly higher expressions of IL6, MMP2, BCL6 and ITGA4 were measured in cells attached to resin-composite surfaces than controls. There were no significant differences between the results using different conditions of resin composite, with or without TEGDMA and well or partially cured. Less cell attachment and presence of more inflammatory markers were observed on all freshly-made resin-composite surfaces. However, after a leakage period attachment of cells to the resin composite improved to the level of natural tooth materials such as enamel. This indicated that the negative effects of resin composites on epithelial cells might be transient.