Three-dimensional (3D) printing is a valuable tool in the production of complexes structures with specific shapes for tissue engineering. Differently from native tissues, the printed structures are static and do not transform their shape in response to different environment changes. Stimuli-responsive biocompatible materials have emerged in the biomedical field due to the ability of responding to other stimuli (physical, chemical, and/or biological), resulting in microstructures modifications. Four-dimensional (4D) printing arises as a new technology that implements dynamic improvements in printed structures using smart materials (stimuli-responsive materials) and/or cells. These dynamic scaffolds enable engineered tissues to undergo morphological changes in a pre-planned way. Stimuli-responsive polymeric hydrogels are the most promising material for 4D bio-fabrication because they produce a biocompatible and bioresorbable 3D shape environment similar to the extracellular matrix and allow deposition of cells on the scaffold surface as well as in the inside. Subsequently, this review presents different bioresorbable advanced polymers and discusses its use in 4D printing for tissue engineering applications.

Resorbable synthetic and natural polymer-based membranes have been extensively studied for guided tissue regeneration. Alloplastic biomaterials are often used for tissue regeneration due to their lower immunoreactivity when compared with allogeneic and xenogeneic materials. Plenum^® Guide is a synthetic membrane material based on polydioxanone (PDO), whose surface morphology closely mimics the extracellular matrix. In this study, Plenum^® Guide was compared with collagen membranes as a barrier material for bone-tissue regeneration in terms of acute and subchronic systemic toxicity. Moreover, characterizations such as morphology, thermal analysis (Tm = 107.35 °C and crystallinity degree = 52.86 ± 2.97 %, final product), swelling (thickness: 0.25 mm ≅ 436% and 0.5 mm ≅ 425% within 24 h), and mechanical tests (E = 30.1 ± 6.25 MPa; σ = 3.92 ± 0.28 MPa; ε = 287.96 ± 34.68%, final product) were performed. The in vivo results revealed that the PDO membranes induced a slightly higher quantity of newly formed bone tissue than the control group (score: treated group = 15, control group = 13) without detectable systemic toxicity (clinical signs and evaluation of the membranes after necropsy did not result in differences between groups, i.e., non-reaction -> tissue-reaction index = 1.3), showing that these synthetic membranes have the essential characteristics for an effective tissue regeneration. Human adipose-derived stem cells (hASCs) were seeded on PDO membranes; results demonstrated efficient cell migration, adhesion, spread, and proliferation, such that there was a slightly better hASC osteogenic differentiation on PDO than on collagen membranes. Hence, Plenum^® Guide membranes are a safe and efficient alternative for resorbable membranes for tissue regeneration.

The extraintestinal pathogen termed avian pathogenic Escherichia coli (APEC) is known to cause colibacillosis in chickens. The molecular basis of APEC pathogenesis is not fully elucidated yet. In this work, we deleted a component of the Yad gene cluster ( yadC ) in order to understand the role of Yad in the pathogenicity of the APEC strain SCI-07. In vitro , the transcription level of yadC was upregulated at 41°C and downregulated at 22°C. The yadC expression in vivo was more pronounced in lungs than in spleen, suggesting a role in the early steps of the infection. Chicks infected with the wild-type and mutant strains presented, respectively, 80% and 50% mortality rates. The Δ yadC strain presented a slightly decreased ability to adhere to HeLa cells with or without the d -mannose analog compared with the wild type. Real-time PCR (RT-PCR) assays showed that fimH was downregulated ( P < 0.05) and csgA and ecpA were slightly upregulated in the mutant strain, showing that yadC modulates expression of other fimbriae. Bacterial internalization studies showed that the Δ yadC strain had a lower number of intracellular bacteria recovered from Hep-2 cells and HD11 cells than the wild-type strain ( P < 0.05). Motility assays in soft agar demonstrated that the Δ yadC strain was less motile than the wild type ( P < 0.01). Curiously, flagellum-associated genes were not dramatically downregulated in the Δ yadC strain. Taken together, the results show that the fimbrial adhesin Yad contributes to the pathogenicity and modulates different biological characteristics of the APEC strain SCI-07.

Avian Pathogenic Escherichia coli is responsible for significant economic losses in the poultry industry by causing a range of systemic or localized diseases collectively termed colibacillosis. The virulence mechanisms of these strains that are pathogenic in poultry and possibly pathogenic in humans have not yet been fully elucidated. This work was developed to study if over-expressed genes in a microarray assay could be potentially involved in the pathogenicity of an Avian Pathogenic Escherichia coli strain isolated from a swollen head syndrome case. For this study, five over-expressed genes were selected for the construction of null mutants [flgE (flagellar hook), tyrR (transcriptional regulator), potF (putrescine transporter), yehD (putative adhesin) and bfr (bacterioferritin)]. The constructed mutants were evaluated for their capacity for the adhesion and invasion of in vitro cultured cells, their motility capacity, and their pathogenic potential in one-day-old chickens compared with the wild-type strain (WT). The Δbfr strain showed a decreased adhesion capacity on avian fibroblasts compared with WT, in the presence and absence of alpha-D-mannopyranoside, and the ΔpotF strain showed decreased adhesion only in the absence of alpha-D-mannopyranoside. The ΔtyrR mutant had a reduced ability to invade Hep-2 cells. No mutant showed changes in invading CEC-32 cells. The mutants ΔflgE and ΔtyrR showed a decreased ability to survive in HD-11 cells. The motility of the mutant strains Δbfr, ΔyehD and ΔpotF was increased, while the ΔtyrR mutant showed reduction, and the ΔflgE became non-motile. No mutant strain caused the same mortality of the WT in one-day-old chickens, showing attenuation to different degrees.

Avian pathogenic Escherichia coli strains cause extraintestinal diseases in birds, leading to substantial economic losses to the poultry industry worldwide. Bacteria that invade cells can overcome the host humoral immune response, resulting in a higher pathogenicity potential. Invasins are members of a large family of outer membrane proteins that allow pathogen invasion into host cells by interacting with specific receptors on the cell surface. An in silico analysis of the genome of a septicemic APEC strain (SEPT362) demonstrated the presence of a putative invasin homologous to the ychO gene from E. coli str. K-12 substr. MG1655. In vitro and in vivo assays comparing a mutant strain carrying a null mutation of this gene, a complemented strain, and its counterpart wild-type strain showed that ychO plays a role in the pathogenicity of APEC strain SEPT362. In vitro assays demonstrated that the mutant strain exhibited significant decreases in bacterial adhesiveness and invasiveness in chicken cells and biofilm formation. In vivo assay indicated a decrease in pathogenicity of the mutant strain. Moreover, transcriptome analysis demonstrated that the ychO deletion affected the expression of 426 genes. Among the altered genes, 93.66 % were downregulated in the mutant, including membrane proteins and metabolism genes. The results led us to propose that gene ychO contributes to the pathogenicity of APEC strain SEPT362 influencing, in a pleiotropic manner, many biological characteristics, such as adhesion and invasion of in vitro cultured cells, biofilm formation and motility, which could be due to the possible membrane location of this protein. All of these results suggest that the absence of gene ychO would influence the virulence of the APEC strain herein studied.