Enhanced Cell Attachment Research Articles

Validation and scalability of homemade polycaprolactone macrobeads grafted with thermo-responsive poly(N-isopropylacrylamide) for mesenchymal stem cell expansion and harvesting.

In this study, polycaprolactone (PCL) macrobeads were prepared by an oil‐in‐water (o/w) emulsion solvent evaporation method with poly(vinyl alcohol) (PVA) as an emulsifier and conjugated to poly(N‐isopropylacrylamide) (PNIPAAm) to be used as cell carriers with noninvasive cell detachment properties (thermo‐response). Following previous studies with PCL‐PNIPAAm carriers, our objectives were to confirm the successful conjugation on homemade macrobeads and to show the advantages of homemade production over commercial beads to control morphological, biological, and fluidization properties. The effects of PCL concentration on the droplet formation and of flow rate and PVA concentration on the size of the beads were demonstrated. The size of the beads, all spherical, ranged from 0.5 to 3.7 mm with four bead categories based on production parameters. The morphology and size of the beads were observed by scanning electron microscopy to show surface roughness enhancing cell attachment and proliferation compared to commercial beads. The functionalization steps with PNIPAAm were then characterized and confirmed by Fourier transform infrared spectroscopy, scanning electron microscopy, and energy dispersion spectroscopy. PNIPAAm‐grafted macrobeads allowed mesenchymal stem cells (MSCs) to spread and grow for up to 21 days. By reducing the temperature to 25°C, the MSCs were successfully detached from the PCL‐PNIPAAm beads as observed with fluorescence microscopy. Furthermore, we validated the scalability potential of both macrobeads production and conjugation with PCL, to produce easily kilograms of thermo‐responsive macrocarriers in a lab environment. This could help moving such approaches towards clinically and industrially relevant processes were cell expansion is needed at very large scale.

Hybrid electrospun scaffolds based on polylactic acid/ PAMAM dendrimer/gemini surfactant for enhancement of synergistic antibacterial ability for biomedical application

Hybrid electrospun scaffolds based on poly (L-lactic acid) (PLLA)/poly (amidoamine) (PAMAM-G2) dendrimer/gemini surfactant were fabricated for the enhancement of synergistic antibacterial activities. The second generation of poly (amidoamine) (PAMAM-G2) and cationic gemini surfactant were utilized to functionalize the optimum electrospun scaffolds. The gelatination process was utilized to improve the wettability of PLLA scaffolds to extend cell attachment and cell proliferation. PLLA nanofibrous scaffolds were characterized by energy dispersion x-ray, scanning electron microscopy images, mechanical properties, water contact angle, Fourier transform infrared spectroscopy, zeta potential and antibacterial assessment. In vitro cell biocompatibility was evaluated by 3-(4, 5- dimethylthiazoyl-2-yl)-2, 5-diphenyltetrazolium bromide assay and morphology of PC-12 cells cultured on hybrid nanofibrous scaffolds and gelatinized ones. The results indicated that the optimum scaffolds could successfully modify the characteristics of PLLA scaffolds leading to much more appropriate physical and chemical properties. In addition, gelatinized nanofibrous scaffolds reveal more wettability enhancing cell attachment and proliferation. Furthermore, using poly (amidoamine) (PAMAM-G2) and gemini surfactant reveals synergetic antibacterial activity due to the competition between both cationic groups of PAMAM and gemini surfactant. Finally, improved cell adhesion and cell viability on modified scaffolds were confirmed. These favorable properties give a chance for these scaffolds to be used in a wide variety of biomedical applications.

In situ hydrothermal growth and in vitro biocompatibility of TiO2 nanowires grown on TiO2 nanoparticle compacts as novel supportive substrates

ABSTRACT In this work, TiO2 nanoparticle (NP) compacts were employed as novel supportive substrates for synthesis of TiO2 nanowires (NWs) with controlled diameter via an alkali hydrothermal route. SEM observations showed that the alkali hydrothermal treatment caused an in situ conversion of TiO2 NPs on the outer surface of the compacts to TiO2 NWs. The diameter of each individual NW could be facilely tailored from 10 nm to 500 nm through adjustment of the hydrothermal temperature and reaction time. Moreover, compared with TiO2 NWs grown on the conventional Ti metals, those grown on the compacts were much more stable in the aqueous solution. In vitro biocompatible evaluation indicates that the as-synthesised TiO2 NWs supported attachment and proliferation of fibroblasts L929 cells, indicating a good biocompatibility. Moreover, TiO2 NWs with small diameter significantly enhanced cell attachment and proliferation. Thus, the as-synthesised TiO2 NWs might have a potential as novel biocompatible tissue regenerative matrix.

Involvement of N-acetylneuraminate cytidylyltransferase in Edwardsiella piscicida pathogenicity

Edwardsiella piscicida is a gram-negative bacterium that causes Edwardsiellosis in cultured fish. Edwardsiellosis is accompanied by symptoms such as skin lesions, hemorrhage, and necrosis in fish organs, which leads to significant economic losses in the aquaculture industry. Recently, we found that bacterial sialoglycoconjugates may be involved in the infectivity of E. piscicida. The more infectious strains of E. piscicida contain more sialic acid in the bacterial body, and the mRNA level of putative CMP-Neu5Ac synthase (css) is upregulated compared to that in the non-pathogenic strain. However, this putative css gene is yet to be cloned, and the involvement of CSS in E. piscicida pathogenicity remains unclear. Here, we cloned and transferred the css gene from E. piscicida into the FPC498 strain. CSS promoted infection in cultured cells originating from different fish species, and enhanced the mortality of E. piscicida-infected zebrafish larvae. CSS enhanced cell attachment and motility in E. piscicida, which differs from the decreased bacterial growth observed with the sialic acid-supplemented M9 medium. Both fractions (chloroform-methanol)-soluble and -insoluble fraction) prepared from E. piscicida pellet exhibited the increment of sialo-conjugates induced by CSS. Further, lectin blotting revealed the increment of Sia α2-3- and α2-6-, but not α2-8-, -linked glycoprotein in CSS-overexpressing E. piscicida. Overall, these findings indicate the physiological significance of CSS and the role of sialylation in E. piscicida pathogenicity.

3D printing of complex architected metamaterial structures by simple material extrusion for bone tissue engineering

Triply periodic minimal surfaces (TPMS) are gaining popularity as scaffolds for bioapplications due to their unique structure, offering strong mechanical properties and biomorphic surfaces which enhance cell attachment and proliferation. In this work, polymer TPMS sheet lattices were printed using a well-known yet unprecedented technique of manufacturing such structures; which is material extrusion (specifically, pneumatic melt extrusion). This method offers a one step, straightforward yet reliable way to print complex porous structures while retaining design accuracy and significantly simplifying the process. Multiple primitive, gyroid and cubic structures were designed using MSLattice and Solidworks with 70% porosity and 2×2×3 unit cells. The scaffolds were printed by melt extrusion of polycaprolactone (PCL) at different parameters to establish the optimal settings. Morphological features (pore size and strut thickness) were determined using scanning electron microscopy (SEM) and the accuracy of print was determined by comparing to the design, showing high print accuracy and minimal percentage errors of less than 15% in all prints. Uniaxial compression testing was used to demonstrate the different deformation processes of the scaffolds and evaluate their mechanical properties, with primitive having the highest modulus and gyroid the highest yield strength. Finally, cell viability was quantified by alamar blue cell viability assay and visualized by SEM, displaying significant increase in cell proliferation and attachment, specifically in the primitive structure. Herein we will explain the challenges faced with design and print optimization and how we overcame them, making this work the first of its kind in material extrusion (pneumatic melt extrusion) printing of TPMS scaffolds.

Preparation and characterization of polyelectrolyte multilayer coatings on 316L stainless steel for antibacterial and bone regeneration applications

A polyelectrolyte multilayer (PEM) coating was fabricated via the alternate deposition of collagen (Col) and poly (γ-glutamic acid) (γ-PGA) onto 316L stainless steel with chitosan as a barrier in the middle layer using spin-assisted layer-by-layer assembly. The γ-PGA layers were loaded with vancomycin (VA) and strontium-containing bioactive glass (SrBG) for antibacterial and bone regeneration applications. The physicochemical properties of the obtained films were characterized by X-ray diffractometry, Fourier-transform infrared spectroscopy, white light interferometry, scanning electron microscopy, water contact angle, hardness, and reduced Young's modulus. The biomineralization process of these films in simulated body fluids (SBF) was assessed. The PEM/VA/SrBG composite films exhibited a smoother and more uniform distribution of particles on the substrate surface. These films exhibited intense surface roughness and good wettability with a contact angle of 58.12°. The PEM/VA/SrBG film showed intensified mechanical properties that may enhance cell attachment and bone regeneration ability. Thus, loading SrBG onto the implant film surface may improve osseointegration activity. The PEM/VA/SrBG film performed intense bioactivity with resulting formation of apatite layer after immersion in SBF. These PEM composite films exhibited antibacterial properties with the release of VA from the film surface. Based on the MTT assay results, the composite films exhibited excellent biocompatibility with bone marrow mesenchymal stem cells. The histology results indicated that the composite films possess enhanced angiogenesis and bone regeneration ability on 21 days that could grow onto the film surface for tissue engineering. Therefore, PEM/VA/SrBG composite films have excellent potential for orthopedic implant applications based on their intense antibacterial, osseointegration, and bone regeneration properties.

Adhesive and biodegradable membranes made of sustainable catechol-functionalized marine collagen and chitosan

We describe bioadhesive membranes developed from marine renewable biomaterials, namely chitosan and collagen extracted from fish skins. Collagen was functionalized with catechol groups (Coll-Cat) to provide the membranes with superior adhesive properties in a wet environment and blended with chitosan to improve the mechanical properties. The blended membranes were compared to chitosan and chitosan blended with unmodified collagen in terms of surface morphology, wettability, weight loss, water uptake, mechanical and adhesive properties. The metabolic activity, the viability and the morphology of L929 fibroblastic cells seeded on these membranes were also assessed. Our results show that the functionalization with catechol groups improves the adhesive and mechanical properties of the membranes and enhances cell attachment and proliferation. These data suggest that the developed marine origin-raw membranes present a potential towards the restoration of the structural and functional properties of damaged soft tissues.

Combination of Polysaccharide Nanofibers Derived from Cellulose and Chitin Promotes the Adhesion, Migration and Proliferation of Mouse Fibroblast Cells.

Extracellular matrix (ECM) as a structural and biochemical scaffold to surrounding cells plays significant roles in cell adhesion, migration, proliferation and differentiation. Herein, we show the novel combination of TEMPO-oxidized cellulose nanofiber (TOCNF) and surface-N-deacetylated chitin nanofiber (SDCtNF), respectively, having carboxylate and amine groups on each crystalline surface, for mouse fibroblast cell culture. The TOCNF/SDCtNF composite scaffolds demonstrated characteristic cellular behavior, strongly depending on the molar ratios of carboxylates and amines of polysaccharide NFs. Pure TOCNF substrate exhibited good cell attachment, although intact carboxylate-free CNF made no contribution to cell adhesion. By contrast, pure SDCtNF induced crucial cell aggregation to form spheroids; nevertheless, the combination of TOCNF and SDCtNF enhanced cell attachment and subsequent proliferation. Molecular blend of carboxymethylcellulose and acid-soluble chitosan made nearly no contribution to cell culture behavior. The wound healing assay revealed that the polysaccharide combination markedly promoted skin repair for wound healing. Both of TOCNF and SDCtNF possessed rigid nanofiber nanoarchitectures with native crystalline forms and regularly-repeated functional groups, of which such structural characteristics would provide a potential for developing cell culture scaffolds having ECM functions, possibly promoting good cellular adhesion, migration and growth in the designated cellular microenvironments.

Poly-L-Lysine and Human Plasmatic Fibronectin Films as Proactive Coatings to Improve Implant Biointegration.

The success of stable and long-term implant integration implies the promotion, control, and respect of the cell microenvironment at the site of implantation. The key is to enhance the implant–host tissue cross talk by developing interfacial strategies that guarantee an optimal and stable seal of soft tissue onto the implant, while preventing potential early and late infection. Indeed, implant rejection is often jeopardized by lack of stable tissue surrounding the biomaterial combined with infections which reduce the lifespan and increase the failure rate of implants and morbidity and account for high medical costs. Thin films formed by the layer-by-layer (LbL) assembly of oppositely charged polyelectrolytes are particularly versatile and attractive for applications involving cell–material contact. With the combination of the extracellular matrix protein fibronectin (Fn, purified from human plasma) and poly-L-lysine (PLL, exhibiting specific chain lengths), we proposed proactive and biomimetic coatings able to guarantee enhanced cell attachment and exhibiting antimicrobial properties. Fn, able to create a biomimetic interface that could enhance cell attachment and promote extracellular cell matrix remodeling, is incorporated as the anionic polymer during film construction by the LbL technic whereas PLL is used as the cationic polymer for its capacity to confer remarkable antibacterial properties.

Chitosan-coated pore wall polycaprolactone three-dimensional porous scaffolds fabricated by porogen leaching method for bone tissue engineering: a comparative study on blending technique to fabricate scaffolds.

One of the significant challenges in the fabrication of scaffolds for tissue engineering lies in the direct interaction of bioactive agents with cells in the scaffolds matrix, which curbs the effectiveness of bioactive agents resulting in diminished cell recognition and attachment ability of the scaffolds. Here, three-dimensional porous scaffolds were fabricated using polycaprolactone (PCL) and chitosan, by two approaches, i.e., blending and surface coating to compare their overall effectiveness. Blended scaffolds (Chi-PCL) were compared with the scaffolds fabricated using surface coating technique, where chitosan was coated on the pore wall of PCL scaffolds (C-PCL). The C-PCL exhibited a collective improvement in bioactivities of the stem cell on the scaffold, because of the cell compatible environment provided by the presence of chitosan over the scaffolds interface. The C-PCL showed the enhanced cell attachment and proliferation behavior of the scaffolds along with two-fold increase in hemolysis compatibility compared to Chi-PCL. Furthermore, the compression strength in C-PCL increased by 24.52% and 8.62% increase in total percentage porosity compared to Chi-PCL was attained. Along with this, all the bone markers showed significant upregulation in C-PCL scaffolds, which supported the surface coating technique over the conventional methods, even though the pore size of C-PCL was compromised by 19.98% compared with Chi-PCL.

Poly(vinyl alcohol)-methacrylate with CRGD peptide: A photocurable biocompatible hydrogel.

Polyvinyl alcohol (PVA)-based hydrogels are promising biomaterials for tissue engineering printing applications. However, one of their main disadvantages is their inability to support cell attachment, which is a critical feature for the preparation of biological scaffolds. The goal of this study was to develop a printable, cell-supportive PVA-based bioink with tunable mechanical properties, without using animal-derived polymers which potentially harbor human pathogens. An ultraviolet light (UV) curable PVA-methacrylate (PVA-MA) polymer mixed with Cys-Arg-Gly-Asp (CRGD) peptide was developed. This peptide holds the integrin receptor binding sequence - RGD, that can enhance cell attachment. The additional cysteine was designed to enable its thiol binding under UV to methacrylate groups of the UV curable PVA-MA. Vero cell, as an adherent cell model was used to assess the hydrogel's cell adhesion. It was found that the PVA-MA-CRGD formula enables the preparation of hydrogels with excellent cell attachment and had even shown superior cell attachment properties relative to added gelatin. Adding hyaluronic acid (HA) as a rheologic modulator enabled the printing of this new formula. Our overall data demonstrates the applicability of this mixture as a bioink for soft tissue engineering such as skin, adipose, liver or kidney tissue.

Effect of Magnesium on Dentinogenesis of Human Dental Pulp Cells.

Introducing therapeutic ions into pulp capping materials has been considered a new approach for enhancing regeneration of dental tissues. However, no studies have been reported on its dentinogenic effects on human dental pulp cells (HDPCs). This study was designed to investigate the effects of magnesium (Mg2+) on cell attachment efficiency, proliferation, differentiation, and mineralization of HDPCs. HDPCs were cultured with 0.5 mM, 1 mM, 2 mM, 4 mM, and 8 mM concentrations of supplemental Mg2+ and 0 mM (control). Cell attachment was measured at 4, 8, 12, 16, and 20 hours. Cell proliferation rate was evaluated at 3, 7, 10, 14, and 21 days. Crystal violet staining was used to determine cell attachment and proliferation rate. Alkaline phosphatase (ALP) activity was assessed using the fluorometric assay at 7, 10, and 14 days. Mineralization of cultures was measured by Alizarin red staining. Statistical analysis was done using multiway analysis of variance (multiway ANOVA) with Wilks' lambda test. Higher cell attachment was shown with 0.5 mM and 1 mM at 16 hours compared to control (P < 0.0001). Cells with 0.5 mM and 1 mM supplemental Mg2+ showed significantly higher proliferation rates than control at 7, 10, 14, and 21 days (P < 0.0001). However, cell proliferation rates decreased significantly with 4 mM and 8 mM supplemental Mg2+ at 14 and 21 days (P < 0.0001). Significantly higher levels of ALP activity and mineralization were observed in 0.5 mM, 1 mM, and 2 mM supplemental Mg2+ at 10 and 14 days (P < 0.0001). However, 8 mM supplemental Mg2+ showed lower ALP activity compared to control at 14 days (P < 0.0001), while 4 mM and 8 mM supplemental Mg2+showed less mineralization compared to control (P < 0.0001). The study indicated that the optimal (0.5–2 mM) supplemental Mg2+ concentrations significantly upregulated HDPCs by enhancing cell attachment, proliferation rate, ALP activity, and mineralization. Magnesium-containing biomaterials could be considered for a future novel dental pulp-capping additive in regenerative endodontics.

Novel composite 3D-printed filament made from fish scale-derived hydroxyapatite, eggshell and polylactic acid via a fused fabrication approach

The great green composites, the three-dimensional (3D) printing filaments containing bio-based polyester, natural hydroxyapatite and eggshell composites, were rationally designed and fabricated. We report herein novel bioactive 3D-printing filaments prepared from fish scale-derived hydroxyapatite (FHAp), polylactic acid (PLA) and a natural antibacterial agent from eggshell (EGS). The preparation of FHAp and EGS from Asian sea bass (Lates calcarifer) scales and chicken EGS, respectively, are also described. The structural, mechanical and thermal properties of the resulting composites were evaluated together with the water contact angle, free-radical scavenging ability and antibacterial effects. Polylactic acid typically exhibits poor free-radical scavenging ability and no antibacterial effect. Incorporating FHAp and EGS as fillers in the processed filament significantly enhanced the functional performance of the PLA composite. The addition of FHAp enhanced cell attachment and proliferation, suggesting osteointegration potential. The addition of EGS containing CaO component provided the antibacterial properties. PLA/EFHAp composites were porous and displayed excellent tensile strength, proliferation of mouse embryonic fibroblasts in vitro, antioxidant properties, water resistance and antibacterial properties. In addition, the developed PLA/FHAp and EFHAp filaments are amenable to large-scale production and implementation in a variety of in vivo implants of bone or other biomedical appliances.

Light-Inducible Spatio-Temporal Control of TLR4 and NF-κB-Gluc Reporter in Human Pancreatic Cell Line.

Augmented Toll-like receptor 4 (TLR4) expression was found in nearly 70% of patients with pancreatic adenocarcinoma, which is correlated with increased tumorigenesis and progression. In this study, we engineered a new light-oxygen-voltage-sensing (LOV) domain-based optogenetic cell line (opto-TLR4 PANC-1) that enables time-resolved activation of the NF-κB and extracellular-signal regulated kinases (ERK)1/2 signalling pathway upon blue light-sensitive homodimerisation of the TLR4-LOV fusion protein. Continuous stimulation with light indicated strong p65 and ERK1/2 phosphorylation even after 24 h, whereas brief light exposure peaked at 8 h and reached the ground level 24 h post-illumination. The cell line further allows a voltage-dependent TLR4 activation, which can be continuously monitored, turned on by light or off in the dark. Using this cell line, we performed different phenotypic cell-based assays with 2D and 3D cultures, with the aim of controlling cellular activity with spatial and temporal precision. Light exposure enhanced cell attachment, the formation and extension of invadopodia, and cell migration in 3D spheroid cultures, but no significant changes in proliferation or viability could be detected. We conclude that the opto-TLR4 PANC-1 cell line is an ideal tool for investigating the underlying molecular mechanisms of TLR4, thereby providing strategies for new therapeutic options.

Development of Si doped nano hydroxyapatite reinforced bilayer chitosan nanocomposite barrier membranes for guided bone regeneration

Guided Bone Regeneration (GBR) is a widely used process for the treatment of periodontal defects to prevent the formation of surrounding soft tissue at the periodontal defect and to provide hard tissue regeneration. Recently GBR designs have focused on the development of resorbable natural polymer-based barrier membranes due to their biodegradability and excellent biocompatibility. The aim of this study is to fabricate a novel bilayer nanocomposite membrane with microporous sublayer composed of chitosan and Si doped nanohydroxyapatite particles (Si-nHap) and chitosan/PEO nanofiber upper layer. Bilayer membrane was designed to prevent epithelial and fibroblastic cell migration and growth impeding bone formation with its upper layer and to support osteogenic cell bioactivity at the defect site with its sublayer. Microporous and nanofiber layers were fabricated by using freeze-drying and electrospinning techniques respectively. The effect of Si-nHap content on the morphological, mechanical and physical properties of the composites were investigated using SEM, AFM, micro-Ct, compression test, water uptake capacity and enzymatic degradation study. Antimicrobial properties of nanocomposite membranes were investigated with tube dilution and disk diffusion methods. In vitro cytotoxicity of bilayer membranes was evaluated. Saos-2 and NIH/3T3 proliferation studies were carried out on each layer. In vitro bioactivity of Saos-2 and NIH/3T3 cells were evaluated with ALP activity and hydroxyproline content respectively. Results showed that Si-nHap incorporation enhanced the mechanical and physical properties as well as controlling biodegradability of the polymer matrix. Besides, Si-nHap loading induced the bioactivity of Saos-2 cells by enhancing cell attachment, spreading and biomineralization on the material surface. Thus, results supported that designed bilayer nanocomposite membranes can be used as a potential biomaterial for guided bone regeneration in periodontal applications.

Articles of Significant Interest in This Issue.

The protein capsid of noroviruses is composed of a protruding (P) domain and a shell that encases the genomic RNA. Bile salts enhance receptor (CD300lf) binding to the mouse norovirus (MNV) P domain and collapse the highly mobile P domain onto the shell surface. Williams et al. (e00176-21) determined the high-resolution cryo-electron microscopy (cryo-EM) structure of the P domain complexed with a neutralizing Fab and demonstrate that bile salts cause allosteric conformational changes that block antibody recognition to the top of the P domain. Therefore, MNV hijacks the host’s own bile to escape antibody-mediated neutralization while enhancing cell attachment.

Surface Characterization and Biocompatibility of Hydroxyapatite Coating on Anodized TiO2 Nanotubes via PVD Magnetron Sputtering.

Hydroxyapatite (HA) coating has received significant attention in the scientific community for the development of implants, and HA coating on titanium oxide (TiO2) nanotubes has shown potential benefits in the improvement of cell proliferation, adhesion, and differentiation. In this study, a HA coating on a TiO2 nanotubular surface was developed to improve the biocompatibility of the titanium (Ti) surface via magnetron sputtering. Scanning electron microscopy (SEM), surface profilometry, and water contact goniometry revealed that HA-coated TiO2 nanotubes influenced the surface roughness (Ra) and hydrophilicity. The XRD and FTIR peaks indicated the presence of crystalline phases of TiO2 (anatase) and HA-coated TiO2 nanotubes after annealing at 500 °C for 120 min. The HA-coated TiO2 nanotubes showed significantly increased Ra and decreased water contact angle (θ) compared to the as-anodized TiO2 nanotubular and bare CP-Ti surfaces. MTS assay using osteoblast-like cells confirmed that the HA-coated TiO2 nanotubular surface provided an enhanced cell attachment and growth when compared to as-anodized TiO2 nanotubular and pure CP-Ti surfaces.

The Genomic and Structural Organization of SARS-CoV-2: A Mutational Perspective

The ongoing pandemic due to the novel SARS-CoV-2 disease (COVID-19) has exerted a great toll on human health. The SARS-CoV-2 is the third most pathogenic human CoV after SARS-CoV-1 and MERS-CoV, which is classified within the genus Betacoronavirus. Though the actual source of its origin and transmission is still unclear, genetic analysis has shown its very close similarity (~96%) with bat SARS-like CoV. SARS-CoV-2 is a spherically-icosahedral virus with a plus-sense single-strand RNA (~30 kb) genome defined into thirteen open reading frames, which encode 2 non-structural polyproteins, 4 structural proteins and 6 accessory proteins. Of its structural proteins the ‘S1’ subunit of spike (S) contains the cellular ACE-2 receptor binding domain (RBD) whereas the ‘S2’ subunit is required for cell membrane fusion. The membrane (M) protein participates in cell-fusion whereas envelope (E) is necessary for virion assembly and morphogenesis. The non-structural polyproteins (pp1a and pp1b) undergo proteolytic processing to produce a total of 16 small proteins, which are involved in mRNA synthesis and replication. Of the accessory proteins (3a, 6, 7a, 7b, 8 and 9b), few are known to modulate host-innate immunity. Interestingly, ‘3b’ is absent in SARS-CoV-2 that significantly differentiates it from other human CoV. Detection of several novel mutations in ‘3a’, ‘3b’ and ‘ORF8’ proteins, notably in the ‘S’ RBD strongly suggest SARS-CoV-2 enhanced cell attachment and facilitated entry, its high infectivity and disease severity in humans. The recent emergence of highly contagious SARS-CoV-2 RBD variants in the United Kingdom (B.1.1.7 strain), South Africa (B.1.351 strain) and Brazil (P.1 strain), and their subsequent spread to other counties have raised serious concerns. Doi: 10.28991/SciMedJ-2021-0301-8 Full Text: PDF

Modulating the physico-mechanical properties of polyacrylamide/gelatin hydrogels for tissue engineering application

Along with providing an environment for cell attachment and proliferation, a tissue engineering scaffolds should possess physical and mechanical properties that would fit the target tissue. The present study aimed to manipulate physico-mechanical properties of polyacrylamide/gelatin hydrogels using response surface method-central composite design (RSM-CCD) to reach a scaffold with defined properties. On this demand, mixtures of gelatin and acrylamide (AAm) monomer were used to prepare semi-interpenetrating hydrogels by free radical polymerization of AAm. Selected variables for statistical modeling were chosen to be weight ratios of monomer/crosslinker, monomer/gelatin, and monomer/initiator. The desired responses were compressive modulus, compressive strength, and swelling. Results showed that desired responses could be tailored by varying these parameters with the highest impact for monomer/crosslinker ratio. The swelling ratio of hydrogels was in the range of 947–1654%, while the modulus varied between 5 and 35 kPa. The cyclic compressive test showed the durability of hydrogels under cyclic loadings. Finally, the results of cell attachment and cytocompatibility analyses indicated that the hydrogels were completely biocompatible and enhanced cell attachment. Thus, these hydrogels could potentially be used as tissue engineering scaffolds for load-bearing organs, including muscle and cartilage, or could be used for in vitro differentiation of stem cells using mechanical clues.

Collagen I Based Enzymatically Degradable Membranes for Organ-on-a-Chip Barrier Models.

Organs-on-chips are microphysiological in vitro models of human organs and tissues that rely on culturing cells in a well-controlled microenvironment that has been engineered to include key physical and biochemical parameters. Some systems contain a single perfused microfluidic channel or a patterned hydrogel, whereas more complex devices typically employ two or more microchannels that are separated by a porous membrane, simulating the tissue interface found in many organ subunits. The membranes are typically made of synthetic and biologically inert materials that are then coated with extracellular matrix (ECM) molecules to enhance cell attachment. However, the majority of the material remains foreign and fails to recapitulate the native microenvironment of the barrier tissue. Here, we study microfluidic devices that integrate a vitrified membrane made of collagen-I hydrogel (VC). The biocompatibility of this membrane was confirmed by growing a healthy population of stem cell derived endothelial cells (iPSC-EC) and immortalized retinal pigment epithelium (ARPE-19) on it and assessing morphology by fluorescence microscopy. Moreover, VC membranes were subjected to biochemical degradation using collagenase II. The effects of this biochemical degradation were characterized by the permeability changes to fluorescein. Topographical changes on the VC membrane after enzymatic degradation were also analyzed using scanning electron microscopy. Altogether, we present a dynamically bioresponsive membrane integrated in an organ-on-chip device with which disease-related ECM remodeling can be studied.