Organotypic in vitro block culture model to investigate tissue-implant interface. An experimental study on pig mandible

In vitro studies of implant-tissue attachment are primarily based on two-dimensional cell culture models, which fail to replicate the three-dimensional native human oral mucosal tissue completely. Thus, the present study aimed to describe a novel tissue culture model using pig mandibular block including alveolar bone and gingival soft tissues to evaluate the tissue attachment to titanium implant provided with hydrothermally induced TiO2 coating. Tissue attachment on TiO2 coated and non-coated implants were compared. Ti-6Al-4V alloy posts were used to function as implants that were inserted in five pig mandibles. Implants were delivered with two different surface treatments, non-coated (NC) titanium and hydrothermal induced TiO2 coated surfaces (HT). The tissue-implant specimens were cultured at an air/liquid interface for 7 and 14 days. The tissue-implant interface was analyzed by histological and immunohistochemical stainings. The microscopic evaluation suggests that pig tissue explants established soft and hard tissue attachment to both implant surfaces. The epithelial cells appeared to attach to the coated implant. The epithelium adjacent to the implant abutment starts to change its phenotype during the early days of the healing process. New bone formation was seen within small pieces of bone in close contact with the coated implant. In conclusion, this in vitro model maintains the viability of pig tissue and allows histologically and immunohistochemically evaluate the tissue-implant interface. HT-induced TiO2 coating seems to have a favorable tissue response. Moreover, this organotypic tissue culture model is applicable for further studies with quantitative parameters to evaluate adhesion molecules present at the implant-tissue interface.

Establishment of a striped catfish skin explant model for studying the skin response in Aeromonas hydrophila infections

Teleost fish skin serves as the first line of defense against pathogens. The interaction between pathogen and host skin determines the infection outcome. However, the mechanism(s) that modulate infection remain largely unknown. A proper tissue culture model that is easier to handle but can quantitatively and qualitatively monitor infection progress may shed some lights. Here, we use striped catfish (Pangasius hypophthalmus) to establish an ex vivo skin explant tissue culture model to explore host pathogen interactions. The skin explant model resembles in vivo skin in tissue morphology, integrity, and immune functionality. Inoculation of aquatic pathogen Aeromonas hydrophila in this model induces epidermal exfoliation along with epithelial cell dissociation and inflammation. We conclude that this ex vivo skin explant model could serve as a teleost skin infection model for monitoring pathogenesis under various infection conditions. The model can also potentially be translated into a platform to study prevention and treatment of aquatic infection on the skin in aquaculture applications.

A genetically encoded tool for reconstituting synthetic modulatory neurotransmission and reconnect neural circuits in vivo

Chemogenetic and optogenetic tools have transformed the field of neuroscience by facilitating the examination and manipulation of existing circuits. Yet, the field lacks tools that enable rational rewiring of circuits via the creation or modification of synaptic relationships. Here we report the development of HySyn, a system designed to reconnect neural circuits in vivo by reconstituting synthetic modulatory neurotransmission. We demonstrate that genetically targeted expression of the two HySyn components, a Hydra-derived neuropeptide and its receptor, creates de novo neuromodulatory transmission in a mammalian neuronal tissue culture model and functionally rewires a behavioral circuit in vivo in the nematode Caenorhabditis elegans. HySyn can interface with existing optogenetic, chemogenetic and pharmacological approaches to functionally probe synaptic transmission, dissect neuropeptide signaling, or achieve targeted modulation of specific neural circuits and behaviors.

Characterization of an Ex Vivo Equine Endometrial Tissue Culture Model Using Next-Generation RNA-Sequencing Technology

Persistent mating-induced endometritis is a major cause of poor fertility rates in the mare. Endometritis can be investigated using an ex vivo equine endometrial explant system which measures uterine inflammation using prostaglandin F2α as a biomarker. However, this model has yet to undergo a wide-ranging assessment through transcriptomics. In this study, we assessed the transcriptomes of cultured endometrial explants and the optimal temporal window for their use. Endometrium harvested immediately post-mortem from native pony mares (n = 8) were sampled (0 h) and tissue explants were cultured for 24, 48 and 72 h. Tissues were stored in RNALater, total RNA was extracted and sequenced. Differentially expressed genes (DEGs) were defined using DESeq2 (R/Bioconductor). Principal component analysis indicated that the greatest changes in expression occurred in the first 24 h of culture when compared to autologous biopsies at 0 h. Fewer DEGs were seen between 24 and 48 h of culture suggesting the system was more stable than during the first 24 h. No genes were differentially expressed between 48 and 72 h but the low number of background gene expression suggested that explant viability was compromised after 48 h. ESR1, MMP9, PTGS2, PMAIP1, TNF, GADD45B and SELE genes were used as biomarkers of endometrial function, cell death and inflammation across tissue culture timepoints. STRING assessments of gene ontology suggested that DEGs between 24 and 48 h were linked to inflammation, immune system, cellular processes, environmental information processing and signal transduction, with an upregulation of most biomarker genes at 24 h. Taken together our observations indicated that 24–48 h is the optimal temporal window when the explant model can be used, as explants restore microcirculation, perform wound healing and tackle inflammation during this period. This key observation will facilitate the appropriate use of this as a model for further research into the equine endometrium and potentially the progression of mating-induced endometritis to persistent inflammation between 24 and 48 h.

Comparison of raloxifene effect on the growth and angiogenesis of human endometrium of healthy and endometriosis subjects: An in vitro three-dimensional tissue culture model

Introduction: Raloxifene (Ral) is the oldest SERM (selective oestrogen receptor modulators) for treatment of breast cancer and osteoporosis. Its oestrogen-modulating effects have been shown in breast and uterus. Since there is little available data on direct Ral effect on the human endometrium, the aim of present study was to investigate the Ral effect on the growth and angiogenesis of the human endometrium of healthy and endometriosis subjects in an in vitro three-dimensional (3D) tissue culture model. Material and methods: Endometrial biopsies from healthy ( n = 9) and endometriosis ( n = 7) patients (endometriotic) were taken and were cut into 1 × 1 mm fragments and implanted between two layers of fibrin jell made by fibrinogen solution (3 mg/ml in medium 199+thrombin). Tissue cultures were performed in 24-wel culture plates. Each biopsy was divided into control wells which received M199 supplemented with FBS (5%) and experimental wells which received same media containing one of raloxifene doses (0.1, 1 and 10 μM). Endometrial tissues were photographed at the beginning and the end of the study period (21 days). Tissue growth and angiogenesis were determined by a scoring system. Results: In control (0), 0.1, 1 and 10 μM Ral, the growth score of normal human endometrial tissues were 1.99, 1.72, 1.53 and 1.12 ( p = 0.02) and angiogenesis percent were 29.6%, 31.28%, 33% and 11.5%. The Growth scores of the endometriotic endometrium were 1.92, 1.82, 1.92 and 1.1 ( p = 0.008) and angiogenesis percent were 36.6%, 16.6%, 44% and 12.5% respectively. Conclusion: Raloxifene showed a different dose dependent effect on endometrial and endometriotic tissue.

Reproducible In Vitro Tissue Culture Model to Study Basic Mechanisms of Calcific Aortic Valve Disease: Comparative Analysis to Valvular Interstitials Cells

The hallmarks of calcific aortic valve disease (CAVD), an active and regulated process involving the creation of calcium nodules, lipoprotein accumulation, and chronic inflammation, are the significant changes that occur in the composition, organization, and mechanical properties of the extracellular matrix (ECM) of the aortic valve (AV). Most research regarding CAVD is based on experiments using two-dimensional (2D) cell culture or artificially created three-dimensional (3D) environments of valvular interstitial cells (VICs). Because the valvular ECM has a powerful influence in regulating pathological events, we developed an in vitro AV tissue culture model, which is more closely able to mimic natural conditions to study cellular responses underlying CAVD. AV leaflets, isolated from the hearts of 6–8-month-old sheep, were fixed with needles on silicon rubber rings to achieve passive tension and treated in vitro under pro-degenerative and pro-calcifying conditions. The degeneration of AV leaflets progressed over time, commencing with the first visible calcified domains after 14 d and winding up with the distinct formation of calcium nodules, heightened stiffness, and clear disruption of the ECM after 56 d. Both the expression of pro-degenerative genes and the myofibroblastic differentiation of VICs were altered in AV leaflets compared to that in VIC cultures. In this study, we have established an easily applicable, reproducible, and cost-effective in vitro AV tissue culture model to study pathological mechanisms underlying CAVD. The valvular ECM and realistic VIC–VEC interactions mimic natural conditions more closely than VIC cultures or 3D environments. The application of various culture conditions enables the examination of different pathological mechanisms underlying CAVD and could lead to a better understanding of the molecular mechanisms that lead to VIC degeneration and AS. Our model provides a valuable tool to study the complex pathobiology of CAVD and can be used to identify potential therapeutic targets for slowing disease progression.

Increased Abundance of Tumour-Associated Neutrophils in HPV-Negative Compared to HPV-Positive Oropharyngeal Squamous Cell Carcinoma Is Mediated by IL-1R Signalling

The incidence of human papillomavirus (HPV)-associated cancer is increasing and HPV is now implicated in the aetiology of more than 60% of all oropharyngeal squamous cell carcinomas (OPSCC). In OPSCC, innate immune cells such as neutrophils and macrophages generally correlate with poor prognosis, whilst adaptive immune cells, such as lymphocytes, tend to correlate with improved prognosis. This may, in part, be due to differences in the immune response within the tumour microenvironment leading to the recruitment of specific tumour-associated leukocyte sub-populations. In this study, we aimed to examine if differences exist in the levels of infiltrated leukocyte sub-populations, with particular emphasis on tumour-associated neutrophils (TAN), and to determine the mechanism of chemokine-induced leukocyte recruitment in HPV-positive compared to HPV-negative OPSCC. Immunohistochemical analysis showed that HPV-negative OPSCC contained significantly more neutrophils than HPV-positive tumours, whilst levels of CD68+ macrophages and CD3+ lymphocytes were similar. Using a 3D tissue culture model to represent tumour-stromal interactions, we demonstrated that HPV-negative tumour-stromal co-cultures expressed significantly higher levels of CXCL8, leading to increased neutrophil recruitment compared to their HPV-positive counterparts. HPV-negative OPSCC cells have previously been shown to express higher levels of IL-1 than their HPV-positive counterparts, indicating that this cytokine may be responsible for driving increased chemokine production in the HPV-negative 3D model. Inhibition of IL-1R in the tumour-stromal models using the receptor-specific antagonist, anakinra, dramatically reduced chemokine secretion and significantly impaired neutrophil and monocyte recruitment, suggesting that this tumour-stromal response is mediated by the IL-1/IL-1R axis. Here, we identify a mechanism by which HPV-negative OPSCC may recruit more TAN than HPV-positive OPSCC. Since TAN are associated with poor prognosis in OPSCC, our study identifies potential therapeutic targets aimed at redressing the chemokine imbalance to reduce innate immune cell infiltration with the aim of improving patient outcome.

Interaction of preimplantation factor with the global bovine endometrial transcriptome

Preimplantation factor (PIF) is an embryo derived peptide which exerts an immune modulatory effect on human endometrium, promoting immune tolerance to the embryo whilst maintaining the immune response to invading pathogens. While bovine embryos secrete PIF, the effect on the bovine endometrium is unknown. Maternal recognition of pregnancy is driven by an embryo-maternal cross talk, however the process differs between humans and cattle. As many embryos are lost during the early part of pregnancy in cattle, a greater knowledge of factors affecting the embryo-maternal crosstalk, such as PIF, is needed to improve fertility. Therefore, for the first time, we demonstrate the effect of synthetic PIF (sPIF) on the bovine transcriptome in an ex vivo bovine endometrial tissue culture model. Explants were cultured for 30h with sPIF (100nM) or in control media. Total RNA was analysed via RNA-sequencing. As a result of sPIF treatment, 102 genes were differentially expressed compared to the control (Padj<0.1), although none by more than 2-fold. The majority of genes (78) were downregulated. Pathway analysis revealed targeting of several immune based pathways. Genes for the TNF, NF-κB, IL-17, MAPK and TLR signalling pathways were down-regulated by sPIF. However, some immune genes were demonstrated to be upregulated following sPIF treatment, including C3. Steroid biosynthesis was the only over-represented pathway with all genes upregulated. We demonstrate that sPIF can modulate the bovine endometrial transcriptome in an immune modulatory manner, like that in the human endometrium, however, the regulation of genes was much weaker than in previous human work.

Modelling persistent Mycoplasma pneumoniae biofilm infections in a submerged BEAS-2B bronchial epithelial tissue culture model

Introduction. Infections with the respiratory pathogen Mycoplasma pneumoniae are often chronic, recurrent and resistant, persisting after antibiotic treatment. M. pneumoniae grown on glass forms protective biofilms, consistent with a role for biofilms in persistence. These biofilms consist of towers of bacteria interspersed with individual adherent cells. Hypothesis/Gap Statement. A tissue culture model for M. pneumoniae biofilms has not been described or evaluated to address whether growth, development and resistance properties are consistent with persistence in the host. Moreover, it is unclear whether the M. pneumoniae cells in the biofilm towers and individual bacterial cells have distinct roles in disease. Aim. We evaluated the properties of biofilms of M. pneumoniae grown on the immortalized human bronchial epithelial cell line BEAS-2B in relation to persistence in the host. We observed nucleation of biofilm towers and the disposition of individual cells in culture, leading to a model of how tower and individual cells contribute to infection and disease. Methodology. With submerged BEAS-2B cells as a substrate, we evaluated growth and development of M. pneumoniae biofilms using scanning electron microscopy and confocal laser scanning microscopy. We characterized resistance to erythromycin and complement using minimum inhibitory concentration assays and quantification of colony forming units. We monitored biofilm tower formation using time-lapse microscopic analysis of host-cell-free M. pneumoniae cultures. Results. Bacteria grown on host cells underwent similar development to those grown without host cells, including tower formation, rounding and incidence of individual cells outside towers. Erythromycin and complement significantly reduced growth of M. pneumoniae . Towers formed exclusively from pre-existing aggregates of bacteria. We discuss a model of the M. pneumoniae biofilm life cycle in which protective towers derive from pre-existing aggregates, and generate individual cytotoxic cells. Conclusion . M. pneumoniae can form protective biofilms in a tissue culture model, implicating biofilms in chronic infections, with aggregates of M. pneumoniae cells being important for establishing infections.