Design and development of 3D printed shape memory triphasic polymer-ceramic bioactive scaffolds for bone tissue engineering.

Scaffolds for bone tissue engineering require considerable mechanical strength to repair damaged bone defects. In this study, we designed and developed mechanically competent composite shape memory triphasic bone scaffolds using fused filament fabrication (FFF) three dimensional (3D) printing. Wollastonite particles (WP) were incorporated into the poly lactic acid (PLA)/polycaprolactone (PCL) matrix as a reinforcing agent (up to 40 wt%) to harness osteoconductive and load-bearing properties from the 3D printed scaffolds. PCL as a minor phase (20 wt%) was added to enhance the toughening effect and induce the shape memory effect in the triphasic composite scaffolds. The 3D-printed composite scaffolds were studied for morphological, thermal, and mechanical properties, in vitro degradation, biocompatibility, and shape memory behaviour. The composite scaffold had interconnected pores of 550 µm, porosity of more than 50%, and appreciable compressive strength (∼50 MPa), which was over 90% greater than that of the pristine PLA scaffolds. The flexural strength was improved by 140% for 40 wt% of WP loading. The inclusion of WP did not affect the thermal property of the scaffolds; however, the inclusion of PCL reduced the thermal stability. An accelerated in vitro degradation was observed for WP incorporated composite scaffolds compared to pristine PLA scaffolds. The inclusion of WP improved the hydrophilic property of the scaffolds, and the result was significant for 40 wt% WP incorporated composite scaffolds having a water contact angle of 49.61°. The triphasic scaffold exhibited excellent shape recovery properties with a shape recovery ratio of ∼84%. These scaffolds were studied for their protein adsorption, cell proliferation, and bone mineralization potential. The incorporation of WP reduced the protein adsorption capacity of the composite scaffolds. The scaffold did not leach any toxic substance and demonstrated good cell viability, indicating its biocompatibility and growth-promoting behavior. The osteogenic potential of the WP incorporated scaffolds was observed in MC3T3-E1 cells, revealing early mineralization in pre-osteoblast cells cultured in different WP incorporated composite scaffolds. These results suggest that 3D-printed WP reinforced PLA/PCL composite bioactive scaffolds are promising for load bearing bone defect repair.

LP30K protein manifested in hemocytes of Bombyx mori larva on Nosema bombycis infection and showed functional evolution based on glucose- binding domain.

Infection by microsporidian Nosema bombycis induced appearance of exclusive protein conjugate of 190 kDa in hemocytes of silkworm Bombyx mori L (Lepidoptera: Bombycidae). Mass spectrometry of the band showed peptides of low molecular weight 30 kDa lipoprotein (LP30K). Six accessions of LP30K identified from the hemocytes comprised 30 K lipoprotein 1, 30 K protein 1, 2, 6, 7 and 11. Two uncharacterised proteins (UCP) identified from the hemocytes showed 100% similarity with LP30K sequence, altogether showed abundance after the infection. The LP30K accessions H9J4F6 (Q00802), E5EVW2 and the UCP accessions D4QGC0 and D4QGB9 showed presence of glucose binding protein I domain "ADSDVPNDILEEQLYNSIVVADYDSAVEK" that binds with fungal glucans to inhibit infection. However glucose binding protein II domain "TLAPRTDDVLAEQLYMSVVIGEYETAIAK" is absent in LP30K accessions from hemocytes showed loss of DNA sequences encoding the domain. The accessions H9J4F5, H9B440, A7LIK7 and H9B444 showed 92% identity with B. mori LP30K protein (NP_001095198.2) however the glucose binding domain I is absent in these accessions suggesting isoform- specific restricted fungal defense activity. Phylogeny tree of the LP30K homologues showed four groups including microvitellogenin and 30 kDa proteins showing functional diversity endorsed with evolutionary diversity. LP30K accessions with glucose binding domain diverged from that without glucose binding domain exemplify co-evolution for domain- dependent functional roles like storage and immune reactions. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03685-x.

Diversity of Antimicrobial Peptides in Silkworm.

Antimicrobial resistance is a phenomenon that the present-day world is witnessing that poses a serious threat to global health. The decline in the development of novel therapeutics over the last couple of decades has exacerbated the situation further. In this scenario, the pursuit of new alternative therapeutics to commonly used antibiotics has gained predominance amongst researchers across the world. Antimicrobial peptides (AMPs) from natural sources have drawn significant interest in the recent years as promising pharmacological substitutes over the conventional antibiotics. The most notable advantage of AMPs is that microorganisms cannot develop resistance to them. Insects represent one of the potential sources of AMPs, which are synthesized as part of an innate immune defence against invading pathogens. AMPs from different insects have been extensively studied, and silkworm is one of them. Diverse classes of AMPs (including attacins, cecropins, defensins, enbocins, gloverins, lebocins and moricins) were identified from silkworm that exhibit antimicrobial property against bacteria, fungi and viruses, indicating their potential therapeutic benefits. This review briefs about the immune responses of silkworm to invading pathogens, the isolation of AMPs from silkworms, AMPs reported in silkworms and their activity against various microorganisms.

Dipteran endoparasitoid Exorista bombycis utilizes antihemocyte components against host defense of silkworm Bombyx mori.

Dipteran endoparasitoids avoid host immune response; however, antidefense components from the Dipterans are unknown. Infestation of commercial silkworm Bombyx mori Linnaeus (Lepidoptera: Bombycidae) by endoparasitoid Exorista bombycis Louis (Diptera: Tachinidae) induced immune reactions, cytotoxicity, granulation, degranulation, and augmented release of cytotoxic marker enzyme lactate dehydrogenase (LDH), and degranulation-mediator enzyme ß-hexosaminidase in hemocytes. In this study, by reverse phase high-performance liquid chromatography, fractions of E. bombycis larval tissue protein with antihemocytic activity are separated. From the fraction, peptides of hemocyte aggregation inhibitor protein (HAIP) and pyridoxamine phosphate oxidase (PNPO) are identified by mass spectrometry. Interacting partners of HAIP and PNPO are retrieved that further enhance the virulence of the parasitoid. PNPO and HAIP genes showed a four- to seven fold increase in expression in the integument of the parasitoid larva. Together, the dipteran endoparasitoid E. bombycis exploit antihemocyte activity to inhibit host defense reactions in addition to defense evasion contemplated.

Molecular insights of metastasis and cancer progression derived using 3D cancer spheroid co-culture in vitro platform.

The multistep metastasis process is carried out by the combinatorial effect of the stromal cells and the cancerous cells and plays vital role in the cancer progression. The scaffold/physical cues aided 3D cancer spheroid imitates the spatiotemporal organization and physiological properties of the tumor. Understanding the role of the key players in different stages of metastasis, the molecular cross-talk between the stromal cells and the cancer cells contributing in the advancement of the metastasis through 3D cancer spheroid co-culture in vitro platform is the center of discussion in the present review. This state-of-art in vitro platform utilized to study the cancer cell host defence and the role of exosomes in the cross talk leading to cancer progression has been critically examined here. 3D cancer spheroid co-culture technique is the promising next-generation in vitro approach for exploring potent treatments and personalized medicines to combat cancer metastasis leading to cancer progression.

Release of Mediator Enzyme ß-Hexosaminidase and Modulated Gene Expression Accompany Hemocyte Degranulation in Response to Parasitism in the Silkworm Bombyx mori.

In insects infections trigger hemocyte-mediated immune reactions including degranulation by exocytosis; however, involvement of mediator enzymes in degranulation process is unknown in insects. We report here that in silkworm Bombyx mori, infection by endoparasitoid Exorista bombycis and microsporidian Nosema bombycis activated granulation in granulocytes and promoted degranulation of accumulated structured granules. During degranulation the mediator lysosomal enzyme ß-hexosaminidase showed increased activity and expression of ß-hexosaminidase gene was enhanced. The events were confirmed in vitro after incubation of uninfected hemocytes with E. bombycis larval tissue protein. On infection, cytotoxicity marker enzyme lactate dehydrogenase (LDH) was released from the hemocytes illustrating cell toxicity. Strong positive correlation (R2 = 0.71) between LDH activity and ß-hexosaminidase released after the infection showed parasitic-protein-induced hemocyte damage and accompanied release of the enzymes. Expression of ß-hexosaminidase gene was enhanced in early stages after infection followed by down regulation. The expression showed positive correlation (R2 = 0.705) with hexosaminidase activity pattern. B. mori hexosaminidase showed 98% amino acid similarity with that of B. mandarina showing origin from same ancestral gene; however, 45-60% varied from other lepidopterans showing diversity. The observation signifies the less known association of hexosaminidase in degranulation of hemocytes induced by parasitic infection in B. mori and its divergence in different species.

The dipteran parasitoid Exorista bombycis induces pro- and anti-oxidative reactions in the silkworm Bombyx mori: Enzymatic and genetic analysis.

Hymenopteran parasitoids inject various factors including polydnaviruses along with their eggs into their host insects that suppress host immunity reactions to the eggs and larvae. Less is known about the mechanisms evolved in dipteran parasitoids that suppress host immunity. Here we report that the dipteran, Exorista bombycis, parasitization leads to pro-oxidative reactions and activation of anti-oxidative enzymes in the silkworm Bombyx mori larva. We recorded increased activity of oxidase, superoxide dismutase, thioredoxin peroxidase, catalase, glutathione-S-transferase (GST), and peroxidases in the hemolymph plasma, hemocytes, and fat body collected from B. mori after E. bombycis parasitization. Microarray and qPCR showed differential expression of genes encoding pro- and anti-oxidant enzymes in the hemocytes. The significance of this work lies in increased understanding of dipteran parasitoid biology.

Antibacterial activity of neem nanoemulsion and its toxicity assessment on human lymphocytes in vitro.

Neem (Azadirachta indica) is recognized as a medicinal plant well known for its antibacterial, antimalarial, antiviral, and antifungal properties. Neem nanoemulsion (NE) (O/W) is formulated using neem oil, Tween 20, and water by high-energy ultrasonication. The formulated neem NE showed antibacterial activity against the bacterial pathogen Vibrio vulnificus by disrupting the integrity of the bacterial cell membrane. Despite the use of neem NE in various biomedical applications, the toxicity studies on human cells are still lacking. The neem NE showed a decrease in cellular viability in human lymphocytes after 24 hours of exposure. The neem NE at lower concentration (0.7-1 mg/mL) is found to be nontoxic while it is toxic at higher concentrations (1.2-2 mg/mL). The oxidative stress induced by the neem NE is evidenced by the depletion of catalase, SOD, and GSH levels in human lymphocytes. Neem NE showed a significant increase in DNA damage when compared to control in human lymphocytes (P<0.05). The NE is an effective antibacterial agent against the bacterial pathogen V. vulnificus, and it was found to be nontoxic at lower concentrations to human lymphocytes.