A review on 3D printing in tissue engineering applications

In tissue engineering, 3D printing is an important tool that uses biocompatible materials, cells, and supporting components to fabricate complex 3D printed constructs. This review focuses on the cytocompatibility characteristics of 3D printed constructs, made from different synthetic and natural materials. From the overview of this article, inkjet and extrusion-based 3D printing are widely used methods for fabricating 3D printed scaffolds for tissue engineering. This review highlights that scaffold prepared by both inkjet and extrusion-based 3D printing techniques showed significant impact on cell adherence, proliferation, and differentiation as evidenced by in vitro and in vivo studies. 3D printed constructs with growth factors (FGF-2, TGF-β1, or FGF-2/TGF-β1) enhance extracellular matrix (ECM), collagen I content, and high glycosaminoglycan (GAG) content for cell growth and bone formation. Similarly, the utilization of 3D printing in other tissue engineering applications cannot be belittled. In conclusion, it would be interesting to combine different 3D printing techniques to fabricate future 3D printed constructs for several tissue engineering applications.

Loss of an Intimin-Like Protein Encoded on a Uropathogenic E. coli Pathogenicity Island Reduces Inflammation and Affects Interactions with the Urothelium

Uropathogenic Escherichia coli (UPEC) causes the majority of uncomplicated urinary tract infections (UTI), which affect nearly half of women worldwide. Many UPEC strains encode an annotated intimin-like adhesin ( ila ) locus in their genome related to a well-characterized virulence factor in diarrheagenic E. coli pathotypes. Its role in UPEC uropathogenesis, however, remains unknown. In prototype UPEC strain CFT073, there is an ila locus that encodes three predicted intimin-like genes sinH , sinI , and ratA . We used in silico approaches to determine the phylogeny and genomic distribution of this locus among uropathogens. We found that the currently annotated intimin-encoding proteins in CFT073 are more closely related to invasin proteins found in Salmonella . Deletion of the individual sinH , sinI , and ratA genes did not result in measurable effects on growth, biofilm formation, or motility in vitro . On average, sinH was more highly expressed in clinical strains during active human UTI than in human urine ex vivo . Unexpectedly, we found that strains lacking this ila locus had increased adherence to bladder cells in vitro , coupled with a decrease in bladder cell invasion and death. The sinH mutant displayed a significant fitness defect in the murine model of ascending UTI including reduced inflammation in the bladder. These data confirmed an inhibitory role in bladder cell adherence to facilitate invasion and inflammation; therefore, the ila locus should be termed invasin-like, rather than intimin-like. Collectively, our data suggest that loss of this locus mediates measurable interactions with bladder cells in vitro and contributes to fitness during UTI.

EDL933 Strains of Escherichia coli O157 can Demonstrate Genetic Diversity and Differential Adherence to Bovine Recto-Anal Junction Squamous Epithelial Cells

Background: Differences between Escherichia coli O157 (O157) strains are well-established with some of these strains being associated with major outbreaks in the US. EDL933 is one such O157 strain that caused a multistate outbreak in 1982 and has since been used as a prototype in various O157-related experiments. Objective: As O157 can readily acquire genetic mutations, we sought to determine if the genetic and phenotypic profiles of EDL933 strains from different sources would be consistent. Methods: We evaluated wild-type O157 strains stocked as EDL933 from three different laboratories, in the strain typing Polymorphic Amplified Typing Sequence (PATS) and the bovine rectal-anal junction squamous epithelial (RSE) cell- and HEp-2 cell- adherence assays. In addition, we also verified if Shiga toxins (Stx), the Locus of Enterocyte Effacement (LEE) or curli fimbriae contributed to the adherence phenotypes observed using mutant and wild-type EDL933 isolates. Results: Our results showed differences in PATS profiles and RSE cell-adherence phenotype, with no influence from the Stx or LEE genes, between EDL933 from different sources. Interestingly, the EDL933 strain that demonstrated the most contrasting diffuse adherence phenotype on RSE cells, EDL933-T, had decreased curli production that may have contributed to this phenotype. Conclusion: Our observations suggest that a comprehensive characterization of bacterial isolates, even if assigned to the same strain type prior to use in experiments, is warranted to ensure consistency and reproducibility of results.

Functionalizable coaxial PLLA/PDLA nanofibers with stereocomplexes at the internal interface

Multifunctionality of electrospun polylactic acid (PLA) nonwovens was generated by the morphological design of nanofibers. Coaxial fibers with a lower number average molar mass Mn PLLA core and a higher Mn PDLA shell form PDLA–PLLA stereocrystals at the interface, induced by annealing. In tensile tests under physiological conditions, the core–shell fibers with higher crystallinity (22% compared to 11–14%) had lower Young’s moduli E (9 ± 1 MPa) and lower elongation at break εb (26 ± 3%) than PDLA alone (E = 31 ± 9 MPa, εb = 80 ± 5%), which can be attributed to simultaneous crystallization and relaxation effects. Gelatin incorporated in the PDLA phase was presented on the outer surface providing a biointerface putatively favorable for cell adherence. Gelatin incorporation did not influence the crystallization behavior but slightly lowered Tg (60 → 54 °C). Employing exclusively polymers established in the clinic, multifunctionality was generated by design. Graphic abstract

Construction of Mycoplasma hyopneumoniae P97 Null Mutants

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia, a world-wide problem in the pig industry. This disease is characterized by a dry, non-productive cough, labored breathing, and pneumonia. Despite years of research, vaccines are marginally effective, and none fully protect pigs in a production environment. A better understanding of the host-pathogen interactions of the M. hyopneumoniae-pig disease, which are complex and involve both host and pathogen components, is required. Among the surface proteins involved in virulence are members of two gene families called P97 and P102. These proteins are the adhesins directing attachment of the organism to the swine respiratory epithelium. P97 is the major ciliary binding adhesin and has been studied extensively. Monoclonal antibodies that block its binding to swine cilia have contributed extensively to its characterization. In this study we use recombination to construct null mutants of P97 in M. hyopneumoniae and characterize the resulting mutants in terms of loss of protein by immunoblot using monoclonal antibodies, ability to bind purified swine cilia, and adherence to PK15 cells. Various approaches to recombination with this fastidious mycoplasma were tested including intact plasmid DNA, single-stranded DNA, and linear DNA with and without a heterologous RecA protein. Our results indicate that recombination can be used to generate site-specific mutants in M. hyopneumoniae. P97 mutants are deficient in cilia binding and PK15 cell adherence, and lack the characteristic banding pattern seen in immunoblots developed with the anti-P97 monoclonal antibody.

The Prevalence of Staphylococcus aureus and the Occurrence of MRSA CC398 in Monkey Feces in a Zoo Park in Eastern China

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the important antibiotic resistant pathogens causing infections in humans and animals. The increasing observation of MRSA in wildlife species has raised the concern of its impact on animal health and the potential of zoonotic transmission. This study investigated the prevalence of S. aureus in fecal samples from non-human primates in a zoo located in Jiangsu, China, in which 6 out of 31 (19.4%) fecal samples, and 2 out of 14 (14.3%) indoor room floor swab samples were S. aureus-positive. The antibiotic susceptibility tests of the eight isolates showed that the two isolates were resistant to both penicillin and cefoxitin, the three isolates were resistant only to penicillin, while three isolates were susceptible to all detected antibiotics. The two isolates resistant to cefoxitin were further identified as MRSA by the presence of mecA. Five different spa types were identified including t034 of two MRSA isolates from Trachypithecus francoisi, t189 of two methicillin-susceptible S. aureus (MSSA) isolates from Rhinopithecus roxellana, t377 of two MSSA isolates from Colobus guereza, and two novel spa types t19488 and t19499 from Papio anubis. Whole genome sequencing analysis showed that MRSA t034 isolates belonged to ST398 clustered in clonal complex 398 (CC398) and carried the type B ΦSa3 prophage. The phylogenetic analysis showed that the two MRSA t034/ST398 isolates were closely related to the human-associated MSSA in China. Moreover, two MRSA isolates contained the virulence genes relating to the cell adherence, biofilm formation, toxins, and the human-associated immune evasion cluster, which indicated the potential of bidirectional transfer of MRSA between monkeys and humans. This study is the first to report MRSA CC398 from monkey feces in China, indicating that MRSA CC398 could colonize in monkey and have the risk of transmission between humans and monkeys.

#17: DNA Methylation in Streptococcus pyogenes Promotes M-Protein Expression and Enhances Epithelial Cell Adherence and Persistence on a Mucosal Surface

Background DNA methylation has been extensively studied as a regulator of gene expression among eukaryotes, but the regulatory role for DNA methylation has been far less studied in bacterial pathogens. Streptococcus pyogenes, or Group A Streptococcus, is an important bacterial pathogen of children. Our group has recently shown that S. pyogenes utilizes DNA methylation at N6-methyladenine (m6A) as a regulatory mechanism, modulating gene transcription and influencing the expression of several genes recognized as potential virulence factors. Our goal was to further explore how DNA methylation impacts virulence properties of S. pyogenes through adherence to epithelial cells and persistence on a mucosal surface. Methods S. pyogenes strains HSC12 (M14), MEW123 (M28), and MEW431 (M4) were modified by in-frame genetic deletion of a 3-gene operon encoding the only Type-I Restriction-Modification locus, resulting in mutant strains lacking the majority of m6A base modifications (ΔRSM). S. pyogenes parent and ΔRSM mutant strains were subjected to transcriptional profiling by RT–PCR and RNA-Seq analysis. Adherence rates of streptococci to D562 human pharyngeal epithelial cells and VKE6E7 human vaginal epithelial cells were assessed. A murine vaginal mucosa colonization model was used to monitor streptococcal mucosal persistence. Results The ΔRSM mutants of all three strains lacked essentially all m6A DNA base modifications by dot-blot with anti-m6A antibody and PacBio™ sequencing with methylation analysis. Transcriptional profiling demonstrated that a limited subset of ~20 genes was strongly down-regulated in all of the ΔRSM mutant strains, most notably genes in the core Mga regulon involved in tissue adherence and evasion of the host immune response, including the M protein (emm gene). The ΔRSM mutants of all 3 strains were attenuated for adherence to human respiratory and vaginal epithelial cells compared with parent strains or complemented mutant strains. The HSC12 and MEW431 ΔRSM mutant strains exhibited significantly decreased bacterial burdens over time compared with parent strains in the murine mucosal carriage model. The bacterial burdens of strain MEW123 and its ΔRSM mutant were not significantly different in the murine mucosal carriage model. Expression of R28, an adhesin specifically promoting adherence to vaginal epithelial cells, was not altered in the MEW123 ΔRSM mutant, which may explain the continued persistence of this strain in the murine model. Conclusions DNA methylation influences gene expression at the transcriptional level in S. pyogenes and affects virulence properties in both in vitro and in vivo models of infection. We report that methylation promotes activation of several important virulence factors, including the M protein and other members of the Mga regulon, and influences epithelial cell adherence and streptococcal persistence on a mucosal surface. DNA methylation appears to be an important contributor to bacterial physiology and pathogenesis. Future work will identify the interaction of m6A base modifications and transcriptional regulatory proteins.

A Novel Trichomonas vaginalis Surface Protein Modulates Parasite Attachment via Protein:Host Cell Proteoglycan Interaction

ABSTRACT Trichomonas vaginalis is a highly prevalent, sexually transmitted parasite which adheres to mucosal epithelial cells to colonize the human urogenital tract. Despite adherence being crucial for this extracellular parasite to thrive within the host, relatively little is known about the mechanisms or key molecules involved in this process. Here, we have identified and characterized a T. vaginalis hypothetical protein, TVAG_157210 (TvAD1), as a surface protein that plays an integral role in parasite adherence to the host. Quantitative proteomics revealed TvAD1 to be ∼4-fold more abundant in parasites selected for increased adherence (MA parasites) than the isogenic parental (P) parasite line. De novo modeling suggested that TvAD1 binds N-acetylglucosamine (GlcNAc), a sugar comprising host glycosaminoglycans (GAGs). Adherence assays utilizing GAG-deficient cell lines determined that host GAGs, primarily heparan sulfate (HS), mediate adherence of MA parasites to host cells. TvAD1 knockout (KO) parasites, generated using CRISPR-Cas9, were found to be significantly reduced in host cell adherence, a phenotype that is rescued by overexpression of TvAD1 in KO parasites. In contrast, there was no significant difference in parasite adherence to GAG-deficient lines by KO parasites compared with wild-type, which is contrary to that observed for KO parasites overexpressing TvAD1. Isothermal titration calorimetric (ITC) analysis showed that TvAD1 binds to HS, indicating that TvAD1 mediates host cell adherence via HS interaction. In addition to characterizing the role of TvAD1 in parasite adherence, these studies reveal a role for host GAG molecules in T. vaginalis adherence. IMPORTANCE The ability of the sexually transmitted parasite Trichomonas vaginalis to adhere to its human host is critical for establishing and maintaining an infection. Yet how parasites adhere to host cells is poorly understood. In this study, we employed a novel adherence selection method to identify proteins involved in parasite adherence to the host. This method led to the identification of a protein, with no previously known function, that is more abundant in parasites with increased capacity to bind host cells. Bioinformatic modeling and biochemical analyses revealed that this protein binds a common component on the host cell surface proteoglycans. Subsequent creation of parasites that lack this protein directly demonstrated that the protein mediates parasite adherence via an interaction with host cell proteoglycans. These findings both demonstrate a role for this protein in T. vaginalis adherence to the host and shed light on host cell molecules that participate in parasite colonization.