Substrate Attachment Research Articles

Interfacial structure and protein incorporation in sparsely tethered phospholipid membranes.

Tethered bilayer lipid membranes (tBLMs) are a robust model system for studying the biophysics of cell membranes, including protein-lipid interactions and membrane dynamics. In this study we describe the structural properties of a novel tBLM platform based on self-assembled monolayers (SAMs) on gold presenting sparsely distributed linear tethers. The interfacial architecture of tBLMs built on two types of alkane tether arrangements, homogeneously distributed short tethers and nanoclustered long tethers, were resolved using neutron reflectometry (NR). A series of tBLM systems was prepared and structurally characterized, with variations in membrane phase (gel and fluid lipids), substrate attachment type (floating and tethered), and electrostatic properties (zwitterionic and negatively charged lipids). Furthermore, the versatility of the tBLM platform was demonstrated by incorporating transmembrane proteins, specifically the outer membrane protein F (OmpF), into the tethered bilayer. Quantitative analyses using NR and quartz crystal microbalance with dissipation monitoring (QCM-D) confirmed successful protein incorporation, with an estimated OmpF volume fraction∼18% within the tBLM. The tBLMs exhibited excellent stability and maintained structural integrity under continuous flow conditions during up to 16-hour NR experiments. Our results highlight the adaptability of this sparse tethering system for creating physiologically relevant membrane models, facilitating precise investigations of membrane-associated processes and protein interactions. The study establishes the potential of this platform for advancing biophysical research on cell membranes and membrane proteins, as well as developing biomimetic systems for analytical and screening applications.

Development of Novel Squid Gladius Biomaterials for Cornea Tissue Engineering.

Cornea tissue engineering is strictly dependent on the development of biomaterials that fulfill the strict biocompatibility, biomechanical, and optical requirements of this organ. In this work, we generated novel biomaterials from the squid gladius (SG), and their application in cornea tissue engineering was evaluated. Results revealed that the native SG (N-SG) was biocompatible in laboratory animals, although a local inflammatory reaction was driven by the material. Cellularized biomaterials (C-SG) demonstrated that the SG provides an adequate substrate for cell attachment and growth, and corneal epithelial cells cultured on this biomaterial were able to express crystallin alpha, a marker for this type of cells. Biomechanical analyses showed that N-SG biomaterials have higher Young modulus and lower traction deformation than control native corneas (CTR), and C-SG showed a similar Young modulus than CTR. Analysis of the optical properties of these samples revealed that the diffuse transmittance of N-SG and C-SG were higher than CTR, with the diffuse reflectance showing the opposite behavior. These results confirm the putative usefulness of this abundant marine-derived biomaterial that can be obtained as a byproduct of the fishing industry.

The role of substrata width and millimeter scale surface micro-structure in the attachment of juvenile mussels

The global mussel aquaculture industry is constrained by the unreliable supply of wild seed mussels and high losses of seed mussels shortly after they are seeded onto coastal farms. The success in the collection of the settling larvae of mussel in the wild and the subsequent on-growing of early seed mussels in aquaculture are largely determined by the settlement and attachment behaviour of the larval and juvenile mussels, which is strongly influenced by the physical structure of their attachment substrata, especially filamentous substrata. This study aimed to identify an ideal set of morphological characteristics of filamentous substrata that would promote the improved attachment of early mussels and contribute to reducing the shortage of seed mussels in the New Zealand green-lipped mussel (Perna canaliculus) aquaculture industry. Artificial filamentous substrata with varying branch width and textured surfaces were tested for their ability to promote the attachment of wild juvenile mussels. The attachment of early juvenile mussels to 1.6 mm wide filamentous substrate was six times higher than their wider counterparts (i.e., 3.7, 5.6, 7.4, and 9.5 mm). There was also a higher proportion of small-size mussels (<0.99 mm shell length) on the 1.6 mm substrata. Furthermore, the filamentous substrata with regularly ridged surface contours (1 mm spacings) tended to attract more juvenile mussels. However, this preference was not consistent for small-size mussels on filaments of thinner width (i.e., 0.4, 0.8, 1.6, and 3.7 mm). Overall, the dimensions of filamentous substrates identified through this study will be valuable for improving the design of artificial substrata used in mussel aquaculture for improving its effectiveness for the attachment of seed mussels.

Ultrastructure of the extraordinary pedal gland in Asplanchna aff. herricki (Rotifera: Monogononta).

Rotifers possess complex morphologies despite their microscopic size and simple appearance. Part of this complexity is hidden in the structure of their organs, which may be cellular or syncytial. Surprisingly, organs that are cellular in one taxon can be syncytial in another. Pedal glands are widespread across Rotifera and function in substrate attachment and/or egg brooding. These glands are normally absent in Asplanchna, which lack feet and toes that function as outlets for pedal glandular secretions in other rotifers. Here, we describe the ultrastructure of a pedal gland that is singular and syncytial in Asplanchna aff. herricki, but is normally paired and cellular in all other rotifers. Asplanchna aff. herricki has a single large pedal gland that is active and secretory; it has a bipartite, binucleate, syncytial body and a cytosol filled with rough endoplasmic reticulum, Golgi, and several types of secretory vesicles. The most abundant vesicle type is large and contains a spherical electron-dense secretion that appears to be produced through homotypic fusion of condensing vesicles produced by the Golgi. The vesicles appear to undergo a phase transition from condensed to decondensed along their pathway toward the gland lumen. Decondensation changes the contents to a mucin-like matrix that is eventually exocytosed in a "kiss-and-run" fashion with the plasma membrane of the gland lumen. Exocytosed mucus enters the gland lumen and exits through an epithelial duct that is an extension of the syncytial integument. This results in mucus that extends from the rotifer as a long string as the animal swims through the water. The function of this mucus is unknown, but we speculate it may function in temporary attachment, prey capture, or floatation.

Morphological, mechanical and biological assessment of PCL/pristine graphene scaffolds for bone regeneration

Scaffolds are physical substrates for cell attachment, proliferation, and differentiation, ultimately leading to the regeneration of tissues. They must be designed according to specific biomechanical requirements such as mechanical properties, surface characteristics, biodegradability, biocompatibility, and porosity. The optimal design of a scaffold for a specific tissue strongly depends on both materials and manufacturing processes. Polymeric scaffolds reinforced with electro-active particles could play a key role in tissue engineering by modulating cell proliferation and differentiation. This paper investigates the use of an extrusion additive manufacturing system to produce PCL/pristine graphene scaffolds for bone tissue applications. PCL/pristine graphene blends were prepared using a melt blending process. Scaffolds with regular and reproducible architecture were produced with different concentrations of pristine graphene. Scaffolds were evaluated from morphological, mechanical, and biological view. The results suggest that the addition of pristine graphene improves the mechanical performance of the scaffolds, reduces the hydrophobicity, and improves cell viability and proliferation.

Snail shell shape, force of attachment, and metabolic rate together cope with the intertidal challenge

Inhabitants of rocky intertidal shores, including gastropods, require specific adaptations to cope with numerous challenges that vary across the intertidal levels. We collected Stramonita biserialis snails from upper (wave-protected and intense predation) and lower (wave-exposed and low predation) intertidal sites to compare the following traits: shell skeleton (ventral and abaxial lateral views of shell shape, thickness, and mass), foot size, energy metabolism, and attachment strength to determine whether the trait values of snails from each zone fit the environmental challenges they face. We used a Principal Component Analysis to reduce the dimensionality of the data. Multivariate Analysis of Covariance (MANCOVA) for comparing characteristics between the two intertidal zones, and Partial Least Squares (PLS) analyses for testing the integration of overall snail characteristics. The traits of the snails of the two intertidal sites matched with the adaptations expected to allow them to cope with their contrasting challenges. The snails from the lower intertidal had more streamlined shells (which reduces drag forces) and a larger aperture and foot extension (which increase the strength of their attachment to the substrate) compared to snails from the upper sites. Snails from the lower intertidal also had a high mass-specific metabolism and soft body proportion, indicating that these snails from the wave-exposed sites have an energetically active musculature that matches their strong substrate attachment. The thin shell walls of the snails of the lower intertidal match the relatively low predatory pressure there.

Enhanced differentiation of mesenchymal stem cells in coral-incorporated PCL/elastin scaffold enables 3D defect healing in osteoporosis rat model

In osteoporosis, bone quality adversely affects the tissue structural competence which increases the risk of a complicated fracture healing. In the present study highly potent scaffold containing natural coral particles was designed and considered for the healing of critical size bone defect in osteoporosis rat model. Scaffold morphological evaluation confirmed the porous nanofibrous structure. Water uptake of about 900 % was obtained for the fabricated scaffold as the result of its composition and three-dimensional structure. Mechanical analysis revealed the compressive modulus of about 50 kPa for the fabricated coral-incorporated nanofibrous structure. In vitro cellular assessments revealed that the designed scaffold induces no toxicity and provides the proper substrate for cell attachment together with increased and prolonged cell proliferation. In vivo experiments demonstrated that implantation of the fabricated scaffold in the femoral defects of osteoporotic rats significantly increased the number of osteocytes and osteoblasts, and enhanced the BTV, and BMP-2 expression compared with the control group. Furthermore, it was observed that seeding the scaffolds with MSCs prior to implantation, resulted in substantial improvements in mRNA expression of the BMP-2 and VEGF genes and considerable enhancement in stereological findings such as significantly higher number of osteoblasts, osteocytes, TVB, and BTV.

Injectable biodegradable microcarriers for iPSC expansion and cardiomyocyte differentiation.

Cell therapy is a potential novel treatment for cardiac regeneration and numerous studies have attempted to transplant cells to regenerate the myocardium lost during myocardial infarction. To date, only minimal improvements to cardiac function have been reported. This is likely to be the result of low cell retention and survival following transplantation. This study aimed to improve the delivery and engraftment of viable cells by using an injectable microcarrier that provides an implantable, biodegradable substrate for attachment and growth of cardiomyocytes derived from induced pluripotent stem cells (iPSC). We describe the fabrication and characterisation of Thermally Induced Phase Separation (TIPS) microcarriers and their surface modification to enable iPSC-derived cardiomyocyte attachment in xeno-free conditions is described. The selected formulation resulted in iPSC attachment, expansion, and retention of pluripotent phenotype. Differentiation of iPSC into cardiomyocytes on the microcarriers is investigated in comparison with culture on 2D tissue culture plastic surfaces. Microcarrier culture is shown to support culture of a mature cardiomyocyte phenotype, be compatible with injectable delivery, and reduce anoikis. The findings from this study demonstrate that TIPS microcarriers provide a supporting matrix for culturing iPSC and iPSC-derived cardiomyocytes in vitro and are suitable as an injectable cell-substrate for cardiac regeneration.

Dislodgement and mortality challenges when restoring shallow mussel beds (Mytilus edulis) in a Danish estuary

In recent decades, mussel beds in the northern Atlantic and Scandinavia have declined rapidly in extent due to anthropogenic impacts, similar to many other marine habitats. In this study, a large‐scale restoration experiment was conducted to identify major challenges that arise during restoration efforts on shallow subtidal mussel beds. Suspension‐grown mussels (Mytilus edulis) were relayed in two different treatments either directly on bare bottom sandy sediments, or on coir nets (Net), used as a proxy for suitable byssal attachment substrate. The treatments were monitored for 1.5 years and coverage (%), biomass (WW), and population dynamics were quantified. Two main challenges of shallow bed restoration were identified: (1) Lack of suitable attachment substrate resulting in dislodgment of individuals during storm events. The Net treatment had significantly higher coverage and biomass of Mytilus at the end of the monitoring period, clearly demonstrating the importance of suitable substrate at physically exposed locations. (2) High mortality of juvenile mussels. Population dynamics revealed a high mortality of juvenile Mytilus, which resulted in almost complete loss of relayed Mytilus individuals less than 30 mm within the first season. This was most likely due to high meso‐predator densities, as a result of declining top‐predator populations. The high mortality of juvenile Mytilus prevented successful annual recruitment, thereby making the population unsustainable long‐term. Both challenges need to be addressed to create stable beds during restoration. Additionally, the experiment demonstrated the viability of using suspension‐grown Mytilus as a seed‐source when restoring mussel beds.

Microbiota characterization of the green mussel Perna viridis at the tissue scale and its relationship with the environment.

Research on the microbiota associated with marine invertebrates is important for understanding host physiology and the relationship between the host and the environment. In this study, the microbiota of the green mussel Perna viridis was characterized at the tissue scale using 16S rRNA gene high-throughput sequencing and compared with the microbiota of the surrounding environment. Different mussel tissues were sampled, along with two environmental samples (the mussel's attachment substratum and seawater). The results showed that the phyla Proteobacteria, Bacteroidetes, and Spirochaetae were dominant in mussel tissues. The bacterial community composition at the family level varied among the tissues of P. viridis. Although the microbiota of P. viridis clearly differed from that of the surrounding seawater, the composition and diversity of the microbial community of the foot and outer shell surface were similar to those of the substratum, indicating their close relationship with the substratum. KEGG prediction analysis indicated that the bacteria harbored by P. viridis were enriched in the degradation of aromatic compounds, osmoregulation, and carbohydrate oxidation and fermentation, processes that may be important in P. viridis physiology. Our study provides new insights into the tissue-scale characteristics of mussel microbiomes and the intricate connection between mussels and their environment.

Improving osseointegration and antimicrobial properties of titanium implants with black phosphorus nanosheets-hydroxyapatite composite coatings for vascularized bone regeneration.

For decades, titanium implants have shown impressive advantages in bone repair. However, the preparation of implants with excellent antimicrobial properties as well as better osseointegration ability remains difficult for clinical application. In this study, black phosphorus nanosheets (BPNSs) were doped into hydroxyapatite (HA) coatings using electrophoretic deposition. The coatings' surface morphology, roughness, water contact angle, photothermal properties, and antibacterial properties were investigated. The BP/HA coating exhibited a surface roughness of 59.1 nm, providing an ideal substrate for cell attachment and growth. The water contact angle on the BP/HA coating was measured to be approximately 8.55°, indicating its hydrophilic nature. The BPNSs demonstrated efficient photothermal conversion, with a temperature increase of 42.2°C under laser irradiation. The BP/HA composite coating exhibited a significant reduction in bacterial growth, with inhibition rates of 95.6% and 96.1% against Staphylococcus aureus and Escherichia coli. In addition, the cytocompatibility of the composite coating was evaluated by cell adhesion, CCK8 and AM/PI staining; the effect of the composite coating in promoting angiogenesis was assessed by scratch assay, transwell assay, and protein blotting; and the osteoinductivity of the composite coating was evaluated by alkaline phosphatase assay, alizarin red staining, and Western blot. The results showed that the BP/HA composite coating exhibited superior performance in promoting biological functions such as cell proliferation and adhesion, antibacterial activity, osteogenic differentiation, and angiogenesis, and had potential applications in vascularized bone regeneration.

Premixed calcium silicate-based ceramic sealers promote osteogenic/cementogenic differentiation of human periodontal ligament stem cells: A microscopy study.

To evaluate the effects of premixed calcium silicate based ceramic sealers on the viability and osteogenic/cementogenic differentiation of human periodontal ligament stem cells (hPDLSCs). The materials evaluated were TotalFill BC Sealer (TFbc), AH Plus Bioceramic Sealer (AHPbc), and Neosealer Flo (Neo). Standardized discs and 1:1, 1:2, and 1:4 eluates of the tested materials were prepared. The following in vitro experiments were carried out: ion release, cell metabolic activity 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell migration, immunofluorescence experiment, cell attachment, gene expression, and mineralization assay. Statistical analyses were performed using one-way ANOVA followed by Tukey's post hoc test (p < .05). Increased Ca2+ release was detected in TFbc compared to AHPbc and Neo (*p < .05). Biological assays showed a discrete cell metabolic activity and cell migration in Neo-treated cell, whereas scanning electronic microscopy assay exhibited that TFbc group had a better cell adhesion process of substrate attachment, spreading, and cytoskeleton development on the niche-like structures of the cement than AHPbc and Neo. The sealers tested were able to induce overexpression of the CEMP-1, ALP, and COL1A1 genes in the first days of exposure, particularly in the case of TFbc (***p < .001). All materials tested significantly increased the mineralization of hPDLSCs when compared to the negative control, although more pronounced calcium deposition was observed in the TFbc-treated cells (***p < .001). Our results suggested that TFbc promotes cell differentiation, both by increasing the expression of key osteo/odontogenic genes and by promoting mineralization of the extracellular matrix, whereas this phenomenon was less evident in Neo and AHPbc. RESEARCH HIGHLIGHTS: TFbc group had a better cell adhesion process of substrate attachment, spreading, and cytoskeleton development on the niche-like structures of the cement than AHPbc and Neo. The sealers tested were able to induce overexpression of the CEMP-1, ALP, and COL1A1 genes in the first days of exposure, particularly in the case of TFbc. All materials tested significantly increased the mineralization of hPDLSCs when compared to the negative control, although more pronounced calcium deposition was observed in the TFbc-treated cells.

A conserved trypanosomatid differentiation regulator controls substrate attachment and morphological development in Trypanosoma congolense.

Trypanosomatid parasites undergo developmental regulation to adapt to the different environments encountered during their life cycle. In Trypanosoma brucei, a genome wide selectional screen previously identified a regulator of the protein family ESAG9, which is highly expressed in stumpy forms, a morphologically distinct bloodstream stage adapted for tsetse transmission. This regulator, TbREG9.1, has an orthologue in Trypanosoma congolense, despite the absence of a stumpy morphotype in that parasite species, which is an important cause of livestock trypanosomosis. RNAi mediated gene silencing of TcREG9.1 in Trypanosoma congolense caused a loss of attachment of the parasites to a surface substrate in vitro, a key feature of the biology of these parasites that is distinct from T. brucei. This detachment was phenocopied by treatment of the parasites with a phosphodiesterase inhibitor, which also promotes detachment in the insect trypanosomatid Crithidia fasciculata. RNAseq analysis revealed that TcREG9.1 silencing caused the upregulation of mRNAs for several classes of surface molecules, including transferrin receptor-like molecules, immunoreactive proteins in experimental bovine infections, and molecules related to those associated with stumpy development in T. brucei. Depletion of TcREG9.1 in vivo also generated an enhanced level of parasites in the blood circulation consistent with reduced parasite attachment to the microvasculature. The morphological progression to insect forms of the parasite was also perturbed. We propose a model whereby TcREG9.1 acts as a regulator of attachment and development, with detached parasites being adapted for transmission.

Engineering of a decellularized bovine skin coated with antibiotics-loaded electrospun fibers with synergistic antibacterial activity for the treatment of infectious wounds.

An ideal antibacterial wound dressing with strong antibacterial behavior versus highly drug-resistant bacteria and great wound-healing capacity is still being developed. There is a clinical requirement to progress the current clinical cares that fail to fully restore the skin structure due to post-wound infections. Here, we aim to introduce a novel two-layer wound dressing using decellularized bovine skin (DBS) tissue and antibacterial nanofibers to design a bioactive scaffold with bio-mimicking the native extracellular matrix of both dermis and epidermis. For this purpose, polyvinyl alcohol (PVA)/chitosan (CS) solution was loaded with antibiotics (colistin and meropenem) and electrospun on the surface of the DBS scaffold to fabricate a two-layer antibacterial wound dressing (DBS-PVA/CS/Abs). In detail, the characterization of the fabricated scaffold was conducted using biomechanical, biological, and antibacterial assays. Based on the results, the fabricated scaffold revealed a homogenous three-dimensional microstructure with a connected pore network, a high porosity and swelling ratio, and favorable mechanical properties. In addition, according to the cell culture result, our fabricated two-layer scaffold surface had a good interaction with fibroblast cells and provided an excellent substrate for cell proliferation and attachment. The antibacterial assay revealed a strong antibacterial activity of DBS-PVA/CS/Abs against both standard strain and multidrug-resistant clinical isolates of Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli. Our bilayer antibacterial wound dressing is strongly suggested as an admirable wound dressing for the management of infectious skin injuries and now promises to advance with preclinical and clinical research.

Animal evolution at the ocean’s water-air interface

Innovation is a key to evolutionary success and entrance into novel ecosystems.1 Species that float freely at the ocean's surface, termed obligate neuston (also called pleuston, here referred to simply as neuston), include highly specialized taxa from distinct evolutionary lineages that evolved floating morphologies.2 In 1958, Soviet scientist, A.I. Savilov,3 stated that floating animal species are derived from benthic ancestors, rather than species from the adjacent pelagic zone, and that floating morphologies are homologous to benthic attachment structures. To test Savilov's hypothesis, we constructed molecular phylogenies and ancestral states for all major floating groups for which molecular data were available. Our results reveal that four of the five clades examined arose directly from a substrate-attached ancestor, although that substrate was not necessarily the benthos, as Savilov stated, and instead included epibiotic and rafting ancestors. Despite their diverse evolutionary origins, floating animals use gas-trapping mechanisms to remain at the surface,4,5,6 and many of these gas-trapping structures appear to be homologous to substrate attachment structures. We also reconstruct the trophic habits of floating mollusks and their sister species, revealing that prey preference remains conserved upon entering the ocean's surface ecosystem. Colonization of the ocean's surface seems to have occurred through successive evolutionary steps from the seafloor. Our results suggest that these steps often included transitions through epibiotic (where species attach to other living organisms) or rafting (where species attach to floating debris) habits. The water-air interface, despite its unique properties, may, in some ways, be just another substrate.

Identification of Mosquito Eggshell Proteins from Aedes aegypti by Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS) Proteomic Analysis.

The insect eggshell is a multifunctional structure with several important roles, including generating an entry point for sperm via the micropyle before oviposition, serving as an oviposition substrate attachment surface, and functioning as a protective layer during embryo development. Eggshell proteins play major roles in eggshell tanning and hardening following oviposition and provide molecular cues that define dorsal-ventral axis formation. Precise eggshell formation during ovarian follicle maturation is critical for normal embryo development and the synthesis of a defective eggshell often gives rise to inviable embryos. Therefore, simple and accurate methods for identifying eggshell proteins will facilitate our understanding of the molecular pathways regulating eggshell formation and the mechanisms underlying normal embryo development. This protocol describes how to isolate and enrich eggshells from mature oocytes of Aedes aegypti mosquitoes and how to extract their eggshell proteins for liquid chromatography with tandem mass spectrometry (LC-MS/MS) proteomic analysis. Although this methodology was developed for studying mosquito eggshells, it may be applicable to eggs from a variety of insects. Mosquitoes are ideal model organisms for this study as their ovarian follicle development and eggshell formation are meticulously regulated by blood feeding and their follicles develop synchronously throughout oogenesis in a time-dependent manner.

Electrospun PAN/PEG Nanofibrous Membrane Embedded with a MgO/gC3N4 Nanocomposite for Effective Bone Regeneration.

Developing biomaterial scaffolds using tissue engineering with physical and chemical surface modification processes can improve the bioactivity and biocompatibility of the materials. The appropriate substrate and site for cell attachment are crucial in cell behavior and biological activities. Therefore, the study aims to develop a conventional electrospun nanofibrous biomaterial using reproducible surface topography, which offers beneficial effects on the cell activities of bone cells. The bioactive MgO/gC3N4 was incorporated on PAN/PEG and fabricated into a nanofibrous membrane using electrospinning. The nanocomposite uniformly distributed on the PAN/PEG nanofiber helps to increase the number of induced pores and reduce the hydrophobicity of PAN. The physiochemical characterization of prepared nanoparticles and nanofibers was carried out using FTIR, X-ray diffraction (XRD), thermogravimetry analysis (TGA), X-ray photoelectron spectroscopy (XPS), and water contact angle measurements. SEM and TEM analyses examined the nanofibrous morphology and the structure of MgO/gC3N4. In vitro studies such as on ALP activity demonstrated the membrane's ability to regenerate new bone and healing capacity. Furthermore, alizarin red staining showed the increasing ability of the cell-cell interaction and calcium content for tissue regeneration. The cytotoxicity of the prepared membrane was about 97.09% of live THP-1 cells on the surface of the MgO/gC3N4@PAN/PEG membrane evaluated using MTT dye staining. The soil burial degradation analysis exhibited that the maximum degradation occurs on the 45th day because of microbial activity. In vitro PBS degradation was observed on the 15th day after the bulk hydrolysis mechanism. Hence, on the basis of the study outcomes, we affirm that the MgO/gC3N4@PAN/PEG nanofibrous membrane can act as a potential bone regenerative substrate.

Degree of cure of orthodontic composite attachments underneath aligners.

The aim of this study was to assess the percentage degree of cure (DC%) of 2-mm-thick resin composite attachments used for aligner treatment. Three types of aligner - two thermoformed aligners (Clear Aligner [CLA], polyethylene terephthalate glycol modified; and Invisalign [INV], polyester urethane) and a three-dimensional-printed aligner (Graphy TC-85DAC [GRP], an acrylate-methacrylate copolymer) - were selected, along with two universal resin composites (3M Filtek Universal [FTU] and Charisma Topaz ONE [CTO]). Samples of each composite were placed under each aligner, and the degree of cure of each composite was evaluated on the top (facing the aligner) and the bottom (facing the substrate) attachment surfaces after curing. Five specimens were used per combination of aligner and composite, and an additional group of composites irradiated without aligners served as the control. The DC% measurements were performed using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The DC% across the aligners were (median values) 33.8%-44.8% for CLA, 33.6%-40.8% for INV, 32.8%-40.6% for GRP, and 40.0%-51.7% for the control group. The DC% values of the attachments cured under any aligner were significantly lower than that of the corresponding control, with the values recorded on the top surfaces being 6% higher than those on the bottom surfaces after adjusting for aligner group and composite type.

The role of vitamin D through SphK1/S1P in the regulation of MS progression

Sphingosine-1-phosphate (S1P) is biologically active lipid, leading to neuroinflammation and macrophage invasion in central nervous system, plays an important role in the development of multiple sclerosis (MS) model in experimental allergic encephalomyelitis (EAE) rats. Vitamin D is observed to be a key factor in regulating cell S1P levels. We detected vitamin D can alleviate the symptoms of EAE rats, but the exact mechanism is unclear. In PC12 cells, vitamin D can reverse S1P-induced cell death, but the signaling pathway unclear. This study was aimed to investigate S1P regulation mechanism or signaling pathway mediated by vitamin D in EAE and PC12 model. In our experiments, S1P and Sphingosine kinase type 1 (SphK1) mRNA and protein expression in EAE rats group, control group, vitamin D feeding group were detected by HPLC, ELISA, RT-PCR and western blot. PC12 cell death was detected by Propidium (PI) staining. VDR plasmid overexpression and RNA interference, immunofluorescence, real-time cell analysis, protein immunoblotting was used to detect SphK1 transcriptional regulation, cell-substrate attachment quality, the signaling pathway of cell apoptosis and inflammation related gene expression (Bax/Bcl-2, Casepase-3, Il-6, TGF-β, TNF-α). Our study showed vitamin D can reverse the elevation of S1P level in EAE rats, reduce the severity and shorten the course of EAE. 1,25-(OH) 2D3 coupled with vitamin D receptor (VDR) inhibited SphK1 transcription. 1,25-(OH)2D3 significantly reduced PC12 cell death rate induced by S1P, in addition improved the cell substrate attachment quality. 1,25-(OH) 2D3 can block S1P-induced p-ERK activation and PI3K /Akt signaling pathway reduced Il-6, TGF-β, TNF-α cytokine release and Bax/Bcl-2, Casepase-3 apoptosis protein expression. On the other hand, immunofluorescence staining showed 1,25-(OH) 2D3 can increase the expression of neuronal perinuclear protein MAP2 in PC12 cells probably protect nerve cells further. In summary, the ameliorative effect of vitamin D was derived from its ability to reduce S1P levels, provides an idea for vitamin D as a combination therapy for disease.

Ligand presentation inside protein crystal nanopores: Tunable interfacial adhesion noncovalently modulates cell attachment

Protein crystals with sufficiently large solvent pores can non-covalently adsorb polymers in the pores. In principle, if these polymers contain cell adhesion ligands, the polymer-laden crystals could present ligands to cells with tunable adhesion strength. Moreover, porous protein crystals can store an internal ligand reservoir, so that the surface can be replenished. In this study, we demonstrate that poly (ethylene glycol) terminated with a cyclic cell adhesion ligand peptide (PEG-RGD) can be loaded into porous protein crystals by diffusion. Through atomic force microscopy (AFM), force-distance correlations of the mechanical interactions between activated AFM tips and protein crystals were precisely measured. The activation of AFM tips allows the tips to interact with PEG-RGD that was pre-loaded in the protein crystal nanopores, mimicking how a cell might attach to and pull on the ligand through integrin receptors. The AFM experiments also simultaneously reveal the detailed morphology of the buffer-immersed nanoporous protein crystal surface. We also show that porous protein crystals (without and with loaded PEG-RGD) serve as suitable substrates for attachment and spreading of adipose-derived stem cells. This strategy can be used to design surfaces that non-covalently present multiple different ligands to cells with tunable adhesive strength for each ligand, and with an internal reservoir to replenish the precisely defined crystalline surface.