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Arthropods from class Arachnida constitute a large and diverse group with over 100,000 described species, and they are sources of many proteins that have a direct impact on human health. Despite the importance of Arachnida, few proteins originating from these organisms have been characterized in terms of their structure. Here we present a detailed analysis of Arachnida proteins that have their experimental structures determined and deposited to the Protein Data Bank (PDB). Our results indicate that proteins represented in the PDB are derived from a small number of Arachnida families, and two-thirds of Arachnida proteins with experimental structures determined are derived from organisms belonging to Buthidae, Ixodidae, and Theraphosidae families. Moreover, 90% of the deposits come from just a dozen of Arachnida families, and almost half of the deposits represent proteins originating from only fifteen different species. In summary, our analysis shows that the structural analysis of proteins originating from Arachnida is not only limited to a small number of the source species, but also proteins from this group of animals are not extensively studied. However, the interest in Arachnida proteins seems to be increasing, which is reflected by a significant increase in the related PDB deposits during the last ten years.

There is a need to develop novel cytocompatible hydrogels for cell encapsulation and delivery in regenerative medicine. The objective of this work was to synthesize isocyanato ethyl methacryloyl-functionalized sericin and determine its material properties as a natural hydrogel for the encapsulation and delivery of human mesenchymal stem cells (MSCs) in regenerative medicine. Sericin extracted from silk cocoons was reacted with 2-isocyanatoethyl methacrylate (IEM) or methacrylic anhydride (MA) to produce sericin urethane methacryloyl (SerAte-UM) or sericin methacryloyl (SerAte-M, control) biopolymers, respectively. The hydrogels produced by photo-crosslinking of the biopolymers in an aqueous solution were characterized with respect to gelation kinetics, microstructure, compressive modulus, water content, degradation, permeability, and viability of encapsulated cells. The secondary structure of citric acid-extracted sericin was not affected by functionalization with IEM or MA. SerAte-UM hydrogel was slightly more hydrophilic than SerAte-M. The gelation time of SerAte-UM hydrogel decreased with an increasing degree of modification. The photo-polymerized SerAte-UM hydrogel had a highly porous, fibrous, honeycomb microstructure with an average pore size in the 40–50 µm range. The compressive modulus, swelling ratio, and permeability of SerAte-UM hydrogel depended on the degree of modification of sericin, and the mass loss after 21 days of incubation in aqueous solution was

Given the great demand for biopolymer and protein-based products from renewable resources, synthesis of a keratin-based hydrogel is presented herein. In this work, a novel hydrogel of poly(γ-glutamic acid) (γ-PGA) and keratin was synthesized through facile EDC·HCl/HOBt chemistry. Since keratin main chain is rich in amino side groups, carboxyl groups in γ-PGA were crosslinked with multi terminated amine groups in keratin. In the following, the hydrogel characteristics, including swelling ratio (2010% at molar ratio of HOBt/EDC = 0.105), in vitro degradation and mass loss (about 20% at day 21 for the aforementioned sample), chemical decomposition and the rheological properties were investigated. The chemical activator agents, enhanced the elastic modulus of swollen hydrogel from around 1000 to 4000 Pa by increasing the crosslinking degree. Despite good biocompatibility for cell growth, some kind of self-assembled keratin hydrogels are not suitable for microscopic observation while the γ-PGA-Keratin hydrogel in our study is transparent. The γ-PGA-Keratin hydrogels possess significant features of rapid hydrogel formation in seconds, maximum swelling ratio of about 2500% maximum elastic modulus (stiffness) of about 4.5 kPa (for the swollen sample) with controllable matrix pore size. For further application, the biocompatibility of the γ-PGA-Keratin hydrogel was assessed by live/dead assay. Recent studies have demonstrated the effect of hydrogel porosity, water absorbing and stiffness on cell spreading, proliferation and differentiation of mesenchymal stem cells. Bone marrow mesenchymal stem cells could be differentiated into various cell fates depending on the elastic modulus of materials they are cultured on. We carried out a statistical study (to skip the cell work labor) to predetermine the proper working span in which we can gain a hydrogel to cover all features needed to be applied for some application like cartilage repair.

IntroductionThe objective was to create multilayer cellular constructs using fetal or adult, decellularized articular cartilage in particulate form as microcarriers for expansion and fusion of mesenchymal stem cells (MSCs) to regenerate the stratified structure of articular cartilage.MethodsPorous microparticles (CMPs) generated from decellularized fetal or adult bovine articular cartilage were used as microcarriers for expansion of human MSCs. The CMP expanded MSCs (CMP-MSCs) were used to generate injectable hydrogels or preformed multilayer constructs for articular cartilage regeneration. In the injectable approach, CMP-MSCs were suspended in alginate gel, crosslinked with calcium chloride, and incubated in chondrogenic medium to generate an injectable regenerative construct. In the preformed approach, fetal or adult CMP-MSCs were suspended in a culture medium, allowed to settle sequentially by the force of gravity, and fused by incubation in chondrogenic medium to generate multilayer cell sheets. The constructs were characterized with respect to compressive modulus, cellularity, and expression of chondrogenic markers.ResultsHuman MSCs expanded on fetal or adult CMPs in basal medium maintained the expression of mesenchymal markers. The injectable CMP-MSCs hydrogels had significantly higher expression of chondrogenic markers and compressive modulus after four weeks incubation in chondrogenic medium compared to MSCs directly encapsulated in alginate gel; preformed CMP-MSCs cell sheets had significantly higher compressive modulus and expression of chondrogenic markers compared to MSCs in the pellet culture.ConclusionThe preformed cell sheet approach is potentially useful for creating multilayer constructs by sequential gravitational settling of CMP-MSCs to mimic the stratified structure of articular cartilage.Insight, Innovation, IntegrationThis work described a novel approach to recreate the zonal structure of articular cartilage. Human MSCs were expanded on porous microcarrier beads generated from decellularized fetal or adult bovine articular cartilage. The cell-seeded microbeads were fused by gravitational settling to form mono- or bi-layer cell sheets. The cell sheets were cultured in chondrogenic medium to regenerate the articular cartilage tissue. The in vitro regenerated tissue had higher compressive modulus and expression of chondrogenic markers compared to the MSC pellet culture.

Scar formation is a common consequence of skin injuries that lead to a wide range of adverse effects including physical deformities and psychological disorders. Studies demonstrate that mammalian fetal skin in early gestation is capable of regenerating itself after injury without any scar formation. A number of potential therapies have been developed to reduce scar formation in cutaneous wounds based on differences between the process of adult and fetal wound healing. The ideal approach to eliminate scar formation after skin injury is to use a pro-regenerative matrix along with growth factors and cell types that induce regeneration rather than repair. This work provides a comprehensive review of engineering approaches to scar-free wound healing with emphasis on the use of pro-regenerative biomaterials to minimize scar formation in skin injuries. IMPACT STATEMENT: Millions of people every year develop scars in response to skin injuries after surgery, trauma, or burns with significant undesired physical and psychological effects. This review provides an update on engineering strategies for scar-free wound healing and discusses the role of different cell types, growth factors, cytokines, and extracellular components in regenerative wound healing. The use of pro-regenerative matrices combined with engineered cells with less intrinsic potential for fibrogenesis is a promising strategy for achieving scar-free skin tissue regeneration.

Engineering approaches that mimic the process of fetal development have the potential to regenerate the zonal organization of articular cartilage. The objective of this study was to investigate the effect of sequential addition of zone-specific growth factors such as bone morphogenetic protein (BMP)-7, insulin-like growth factor (IGF)-1, and Indian hedgehog (IHH) to transforming growth factor (TGF)-β1-supplemented chondrogenic medium on zonal differentiation of human mesenchymal stem cells (hMSCs) encapsulated in an articular cartilage-derived matrix. First, fetal or adult bovine articular cartilage was decellularized, digested, and methacrylate functionalized to produce an injectable macromer (CarMa, f-CarMa for fetal, a-CarMa for adult) for encapsulation of hMSCs. Next, the optimum matrix source and initial cell density for chondrogenic differentiation of hMSCs to the superficial and calcified zone phenotypes were determined by encapsulation of the cells in CarMa hydrogel and incubated in chondrogenic medium/TGF-β1 supplemented with BMP-7 and IHH, respectively. Then, the encapsulated hMSCs were preexposed to BMP-7-supplemented chondrogenic medium/TGF-β1 and the effect of sequential addition of IGF-1 and IHH to the medium on the expression of zone-specific markers was investigated. According to the results, f-CarMa and high cell density enhanced differentiation of the encapsulated hMSCs to the superficial zone phenotype, whereas a-CarMa and low cell density enhanced differentiation to the calcified zone. The addition of IGF-1 to the chondrogenic medium/TGF-β1 stimulated differentiation of the encapsulated hMSCs, preexposed to BMP-7, to the middle zone phenotype. The addition of IHH to the chondrogenic medium/TGF-β1 stimulated maturation of the encapsulated hMSCs, preexposed to BMP-7 and IGF-1, to the calcified zone phenotype. The results are potentially useful for engineering injectable, cellular hydrogels for regeneration of full-thickness articular cartilage. IMPACT STATEMENT: The higher regenerative capacity of fetal articular cartilage compared with the adult is rooted in differences in cell density and matrix composition. We hypothesized that the zonal organization of articular cartilage can be engineered by encapsulation of mesenchymal stem cells in a single superficial zone-like matrix followed by sequential addition of zone-specific growth factors within the matrix, similar to the process of fetal cartilage development. The results demonstrate that the zonal organization of articular cartilage can potentially be regenerated using an injectable, monolayer cell-laden hydrogel with sequential release of growth factors.

In this study the synthesis of bi-phase nanoparticles of Fe1.46Zn0.5La0.04Cu0.5O4 (FZLCs) ceramics were first carried out by Sol-gel method and then nanocomposites of FZLCs with polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), polyethylene glycol (PEG) and polyethylene oxide (PEO) were prepared by one-pot blending technique. XRD, FT-IR, TG/DTA and SEM techniques were applied for complete characterization of composites. Rheological and dielectric properties of all nanocomposites were studied in detail for their comparative performance. TGA results reveal the highly thermal stability for all nanocomposites in this order i.e. FLZCs/PEO > FLZCs/PVA > FZLCs/PVP > FZLCs/PEG. Rheological properties show that these materials are rigid, pseudo plastic and non-Newtonian in nature. The increase in values for storage modulus (G′) and loss modulus (G′′) with increasing angular frequency owed to the shear thinning behavior of these nanocomposites. Dielectric properties show good agreement to that of energy storage substances which means that these materials have potential to be applied in storage devices.

Nonsteroidal anti-inflammatory hydrophobic drugs (NSAIDs) are amongst the most commonly given categories of drugs worldwide in the treatment of pain, irritation and some of them even fever in many conditions. Critical micelle concentration of NSAIDs with Naproxen sodium (NS) and its mixture with non-ionic surfactants i.e. Tween 20, Tween 40 and Tween 60 was investigated at 293 K, 303 K, 313 K and 323 K at different concentrations using surface tension and dynamic laser light scattering (DLS) techniques. Due to amphiphilic behaviour in aqueous solution NS form aggregates at sufficiently high concentration. Thermodynamic/adsorption properties like free energy of micellization (ΔGmic), enthalpy of micellization (ΔHmic), entropy of micellization (ΔSmic), Γmic and area per molecule (A2) of NS in the presence of surfactants were also measured at different temperatures. The results showed that the presence of surfactants favoured the ΔGmic and become more enhanced with increase in temperature. Further the solubility of drug is more favourable with increase in polyethylene chain in basic surfactant molecules i.e. 20–60, which indicates that Tween-60 enhanced the solubility of NS more comparatively to Tween-40 and Tween-20 and may be applied as best additive for solubilisation of NS.

The objective of this work was to engineer self-assembled nanoparticles (NPs) for on-demand release of bone morphogenetic protein-2 (BMP2) and vascular endothelial growth factor (VEGF) in response to enzymes secreted by the migrating human mesenchymal stem cells (hMSCs) and human endothelial colony forming cells (ECFCs) to induce osteogenesis and vasculogenesis. Gene expression profiling experiments revealed that hMSCs and ECFCs, encapsulated in osteogenic/vasculogenic hydrogels, expressed considerable levels of plasminogen, urokinase plasminogen activator and its receptor uPAR, and tissue plasminogen activator. Therefore, the plasmin-cleavable lysine-phenylalanine-lysine-threonine (KFKT) was used to generate enzymatically cleavable NPs. The acetyl-terminated, self-assembling peptide glycine-(phenylalanine)3GFFF-ac and the plasmin-cleavable GGKFKTGG were reacted with the cysteine-terminated CGGK(Fmoc/MTT) peptide through the MTT and Fmoc termini, respectively. The difunctional peptide was conjugated to polyethylene glycol diacrylate (PEGDA) with molecular weights (MW) ranging from 0.5 to 7.5 kDa, and the chain ends of the PEG-peptide conjugate were terminated with succinimide groups. After self-assembly in aqueous solution, BMP2 was grafted to the self-assembled, plasmin-cleavable PEG-based (PxSPCP) NPs for on-demand release. The NPs' stability in aqueous solution and that of the grafted BMP2 were strongly dependent on PEG MW. P2SPCP NPs showed high particle size stability, BMP2 grafting efficiency, grafted protein stability, and high extent of osteogenic differentiation of hMSCs. The localized and on-demand release of BMP2 from PxSPCP NPs coencapsulated with hMSCs in the linear polyethylene glycol-co-lactide acrylate patterned hydrogel with microchannels encapsulating hMSCs + ECFCs and VEGF-conjugated nanogels resulted in the highest extent of osteogenic and vasculogenic differentiation of the encapsulated cells compared to directly added BMP2/VEGF. The on-demand release of BMP2 from PxSPCP NPs not only enhances osteogenesis and vasculogenesis but also potentially reduces many undesired side effects of BMP2 therapy in bone regeneration.

Resveratrol is a small molecule produced by various plants with a remarkable range of beneficial functions in animals. One of these is stimulating signaling pathways in adipose tissue that protect against obesity. Unfortunately, resveratrol suffers from poor bioavailability that inhibits its accumulation in target tissues, including fat, thus hindering the realization of its therapeutic potential. To address this, we are developing biodegradable microparticles as drug depots for controlled release of resveratrol within fat. In this study, resveratrol was encapsulated into poly(lactide-co-glycolide) microparticles using an oil-in-water emulsion/solvent evaporation technique. The oil phase consisted of resveratrol and poly(lactide-co-glycolide) dissolved in a mixture of dichloromethane and ethanol; meanwhile, the aqueous phase contained poly(vinyl alcohol) as the emulsifier. Increasing ethanol’s volume ratio increased resveratrol’s solubility in the oil phase and particle drug loading. The maximal loading achieved was 65 µg/mg (6.5%) and occurred when the ethanol to dichloromethane ratio was 1:3. Under these conditions, particles exhibited ruffled surfaces, which resulted in variable drug release over the first three days of a six-week release assay. By decreasing resveratrol and ethanol in the oil phase and increasing poly(vinyl alcohol) in the aqueous phase, smooth particles were achieved, but they suffered a 15–25-fold decrease in drug loading depending on size. Small particles exhibited higher drug loading and burst drug release compared to larger particles because of their higher specific surface area. Utilizing mild chemistry, we functionalized poly(vinyl alcohol) with fluorescein isothiocyanate and demonstrated that encapsulation of resveratrol in the particle decreases the amount of fluorescent polymer on the particle surface, suggesting resveratrol displaces the emulsifier during particle formation. Taken together, resveratrol can be encapsulated into poly(lactide-co-glycolide) microparticles, but it accumulates at the particle surface impacting drug loading, surface roughness, and drug release.

The objective of this work was to fabricate a rigid, resorbable and osteoconductive scaffold by mimicking the hierarchical structure of the cortical bone. Aligned peptide-functionalize nanofiber microsheets were generated with calcium phosphate (CaP) content similar to that of the natural cortical bone. Next, the CaP-rich fibrous microsheets were wrapped around a microneedle to form a laminated microtube mimicking the structure of an osteon. Then, a set of the osteon-mimetic microtubes were assembled around a solid rod and the assembly was annealed to fuse the microtubes and form a shell. Next, an array of circular microholes were drilled on the outer surface of the shell to generate a cortical bone-like scaffold with an interconnected network of Haversian- and Volkmann-like microcanals. The CaP content, porosity and density of the bone-mimetic microsheets were 240 wt%, 8% and 1.9 g/ml, respectively, which were close to that of natural cortical bone. The interconnected network of microcanals in the fused microtubes increased permeability of a model protein in the scaffold. The cortical scaffold induced osteogenesis and vasculogenesis in the absence of bone morphogenetic proteins upon seeding with human mesenchymal stem cells and endothelial colony-forming cells. The localized and timed-release of morphogenetic factors significantly increased the extent of osteogenic and vasculogenic differentiation of human mesenchymal stem cells and endothelial colony-forming cells in the cortical scaffold. The cortical bone-mimetic nature of the cellular construct provided balanced rigidity, resorption rate, osteoconductivity and nutrient diffusivity to support vascularization and osteogenesis.

The present communication reports on changes in the secondary and tertiary structures of Hemoglobin (Hb) upon fructation. The presence of Lemon balm (LB) provided significant protection toward the formation of early (HbA1c) and advanced glycation end products (Hb-AGEs), as evident from circular dichroism (CD) and fluorescence studies. The degree of AGE modification and its inhibition were characterized with intrinsic fluorescence, the fibrillar state, and the exposure of hydrophobic clusters. An increased signal of intrinsic fluorescence was observed upon glycation, particularly in the later stage of fructation. Glycation mediated fibril formation and its inhibition were assessed by Thioflavin T (ThT) assay. These sequence of events were parallel with an increase in β-sheet content from ~2 to 13% for Hb-AGE, as evidenced by Congo red binding and CD. Furthermore, the late stage of glycation was also marked by an increase loss of heme moiety, confirming the relatively high affinity of ANS toward glycated-globin which was mitigated by applying the balm extract. However, the activity of balm can be attributed to inhibition of oxidative stress and free radicals derived from Hb-AGE, or chelation of metal ions producing in Fenton reaction. Overall, the presence of LB provided significant protection against AGE-induced deleterious processes, can qualify the herb as an effective AGE-inhibitor with potential prevention toward diabetic complications arising from Hb glycation.

The objective of this work was to synthesize an injectable and photopolymerizable hydrogel based on keratin extracted from poultry feather for encapsulation and delivery of stem cells in tissue regeneration. Since feather keratin is rich in cysteine residue, allylation of sulfhydryl groups was used for functionalization of keratin. Keratin was extracted from feather barbs by reducing the disulfide bonds in cysteine residues to sulfhydryl groups (-SH). Next, the free thiol groups were converted to dehydroalanine (Dha) by oxidative elimination using O-(2,4,6-trimethylbenzenesulfonyl) hydroxylamine. Then, the Dha moieties were converted to s-allyl cysteine by reaction with allyl mercaptan to produce keratin allyl thioether (KeratATE) biopolymer. Human mesenchymal stem cell (hMSCs) were suspended in the aqueous solution of KeratATE, injected into a mold, and photopolymerized to generate a KeratATE hydrogel encapsulating hMSCs. The freeze-dried photo-cross-linked KeratATE hydrogels had a porous, interconnected, honeycomb microstructure with pore sizes in the 20-60 μm range. The compressive modulus of the hydrogels ranged from 1 to 8 kPa depending on KeratATE concentration. KeratATE hydrogels had5% mass loss in collagenase solution after 21 days of incubation, whereas the mass loss was 15% in trypsin solution. Degradation of KeratATE hydrogel was strongly dependent on trypsin concentration but independent of collagenase. hMSCs proliferated and adopted an elongated spindle-shape morphology after seeding on KeratATE hydrogel. KeratATE hydrogel supported differentiation of the encapsulated hMSCs to the osteogenic and chondrogenic lineages to the same extent as those hMSCs encapsulated in gelatin methacryloyl hydrogel. The results suggest that keratin allyl thioether hydrogel with controllable degradation is a viable matrix for encapsulation and delivery of stem cells in tissue regeneration.

The objective of this work was to investigate the effect of devitalized human mesenchymal stem cells (hMSCs) and endothelial colony-forming cells (ECFCs) seeded on mineralized nanofiber microsheets on protein release, osteogenesis, vasculogenesis, and macrophage polarization. Calcium phosphate nanocrystals were grown on the surface of aligned, functionalized nanofiber microsheets. The microsheets were seeded with hMSCs, ECFCs, or a mixture of hMSCs+ECFCs, cultured for cell attachment, differentiated to the osteogenic or vasculogenic lineage, and devitalized by lyophilization. The release kinetic of total protein, bone morphogenetic protein-2 (BMP2), and vascular endothelial growth factor (VEGF) from the devitalized microsheets was measured. Next, hMSCs and/or ECFCs were seeded on the devitalized cell microsheets and cultured in the absence of osteo-/vasculo-inductive factors to determine the effect of devitalized cell microsheets on hMSC/ECFC differentiation. Human macrophages were seeded on the microsheets to determine the effect of devitalized cells on macrophage polarization. Based on the results, devitalized undifferentiated hMSC and vasculogenic-differentiated ECFC microsheets had highest sustained release of BMP2 and VEGF, respectively. The devitalized hMSC microsheets did not affect M2 macrophage polarization while vascular-differentiated, devitalized ECFC microsheets did not affect M1 polarization. Both groups stimulated higher M2 macrophage polarization compared to M1.