Genetic and Functional Characterization of a Salicylate 1-monooxygenase Located on an Integrative and Conjugative Element (ICE) in Pseudomonas stutzeri AJR13

Ivanovski Igor,Zylstra Gerben J. 한국미생물학회 2023 The journal of microbiology Vol.61 No.12

Pseudomonas stutzeri strain AJR13 was isolated for growth on the related compounds biphenyl (BPH) and diphenylmethane (DPM). The BPH and DPM degradative pathway genes are present on an integrative and conjugative element (ICE) in the chromosome. Examination of the genome sequence of AJR13 revealed a gene encoding a salicylate 1-monooxygenase (salA) associated with the ICE even though AJR13 did not grow on salicylate. Transfer of the ICE to the well-studied Pseudomonas putida KT2440 resulted in a KT2440 strain that could grow on salicylate. Knockout mutagenesis of the salA gene on the ICE in KT2440 eliminated the ability to grow on salicylate. Complementation of the knockout with the cloned salA gene restored growth on salicylate. Transfer of the cloned salA gene under control of the lac promoter to KT2440 resulted in a strain that could grow on salicylate. Heterologous expression of the salA gene in E. coli BL21 DE3 resulted in the production of catechol from salicylate, confirming that it is indeed a salicylate 1-monooxygenase. Interestingly, transfer of the cloned salA gene under control of the lac promoter to AJR13 resulted in a strain that could now grow on salicylate, suggesting that gene expression for the downstream catechol pathway is intact.

Role of offset and gradient architectures of 3-D melt electrowritten scaffold on differentiation and mineralization of osteoblasts

Naghmeh Abbasi,Saso Ivanovski,Karan Gulati,Robert M. Love,Stephen Hamlet 한국생체재료학회 2020 생체재료학회지 Vol.24 No.1

Background: Cell-scaffold based therapies have the potential to offer an efficient osseous regenerative treatment and PCL has been commonly used as a scaffold, however its effectiveness is limited by poor cellular retention properties. This may be improved through a porous scaffold structure with efficient pore arrangement to increase cell entrapment. To facilitate this, melt electrowriting (MEW) has been developed as a technique able to fabricate cell-supporting scaffolds with precise micro pore sizes via predictable fibre deposition. The effect of the scaffold’s architecture on cellular gene expression however has not been fully elucidated. Methods: The design and fabrication of three different uniform pore structures (250, 500 and 750 μm), as well as two offset scaffolds with different layout of fibres (30 and 50%) and one complex scaffold with three gradient pore sizes of 250–500 - 750 μm, was performed by using MEW. Calcium phosphate modification was applied to enhance the PCL scaffold hydrophilicity and bone inductivity prior to seeding with osteoblasts which were then maintained in culture for up to 30 days. Over this time, osteoblast cell morphology, matrix mineralisation, osteogenic gene expression and collagen production were assessed. Results: The in vitro findings revealed that the gradient scaffold significantly increased alkaline phosphatase activity in the attached osteoblasts while matrix mineralization was higher in the 50% offset scaffolds. The expression of osteocalcin and osteopontin genes were also upregulated compared to other osteogenic genes following 30 days culture, particularly in offset and gradient scaffold structures. Immunostaining showed significant expression of osteocalcin in offset and gradient scaffold structures. Conclusions: This study demonstrated that the heterogenous pore sizes in gradient and fibre offset PCL scaffolds prepared using MEW significantly improved the osteogenic potential of osteoblasts and hence may provide superior outcomes in bone regeneration applications.

In vivo bone regeneration assessment of offset and gradient melt electrowritten (MEW) PCL scaffolds

Naghmeh Abbasi,Ryan S. B. Lee,Saso Ivanovski,Robert M. Love,Stephen Hamlet 한국생체재료학회 2020 생체재료학회지 Vol.24 No.4

Background: Biomaterial-based bone tissue engineering represents a promising solution to overcome reduced residual bone volume. It has been previously demonstrated that gradient and offset architectures of threedimensional melt electrowritten poly-caprolactone (PCL) scaffolds could successfully direct osteoblast cells differentiation toward an osteogenic lineage, resulting in mineralization. The aim of this study was therefore to evaluate the in vivo osteoconductive capacity of PCL scaffolds with these different architectures. Methods: Five different calcium phosphate (CaP) coated melt electrowritten PCL pore sized scaffolds: 250 μm and 500 μm, 500 μm with 50% fibre offset (offset.50.50), tri layer gradient 250–500-750 μm (grad.250top) and 750–500-250 μm (grad.750top) were implanted into rodent critical-sized calvarial defects. Empty defects were used as a control. After 4 and 8 weeks of healing, the new bone was assessed by micro-computed tomography and immunohistochemistry. Results: Significantly more newly formed bone was shown in the grad.250top scaffold 8 weeks postimplantation. Histological investigation also showed that soft tissue was replaced with newly formed bone and fully covered the grad.250top scaffold. While, the bone healing did not happen completely in the 250 μm, offset.50.50 scaffolds and blank calvaria defects following 8 weeks of implantation. Immunohistochemical analysis showed the expression of osteogenic markers was present in all scaffold groups at both time points. The mineralization marker Osteocalcin was detected with the highest intensity in the grad.250top and 500 μm scaffolds. Moreover, the expression of the endothelial markers showed that robust angiogenesis was involved in the repair process. Conclusions: These results suggest that the gradient pore size structure provides superior conditions for bone regeneration.

Release of lithium from 3D printed polycaprolactone scaffolds regulates macrophage and osteoclast response

Bartnikowski, Michal,Moon, Ho-Jin,Ivanovski, Saš,o IOP Publishing 2018 Biomedical materials Vol.13 No.6

Titania nanopores with dual micro-/nano-topography for selective cellular bioactivity

Gulati, Karan,Moon, Ho-Jin,Li, Tao,Sudheesh Kumar, P.T.,Ivanovski, Saš,o Elsevier 2018 Materials Science and Engineering C Vol.91 No.-

<P><B>Abstract</B></P> <P>This letter describes a simple surface modification strategy based on a single-step electrochemical anodization towards generating dual micro- and nano-rough horizontally-aligned TiO<SUB>2</SUB> nanopores on the surface of clinically utilized micro-grooved titanium implants. Primary macrophages, osteoblasts and fibroblasts were cultured on the nano-engineered implants, and it was demonstrated that the modified surfaces selectively reduced the proliferation of macrophages (immunomodulation), while augmenting the activity of osteoblasts (osseo-integration) and fibroblasts (soft-tissue integration). Additionally, the mechanically robust nanopores also stimulated osteoblast and fibroblast adhesion, attachment and alignment along the direction of the pores/grooves, while macrophages remained oval-shaped and sparsely distributed. This study for the first time reports the use of cost-effectively prepared nano-engineered titanium surface via anodization, with aligned multi-scale micro/nano features for selective cellular bioactivity, without the use of any therapeutics.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Single-step fabrication of dual micro-rough and nanoporous titanium surfaces </LI> <LI> Mechanically robust aligned nanopores with preserved underlying micro-features </LI> <LI> Upregulated attachment and adhesion of osteoblasts and fibroblasts </LI> <LI> Reduced macrophage proliferation and adherence </LI> <LI> Osteoblasts and fibroblasts align parallel to the nanopore arrangement. </LI> </UL> </P>

Rapid fabrication of highly porous and biocompatible composite textile tubular scaffold for vascular tissue engineering

Abdal-hay, Abdalla,Memic, Adnan,Hussein, Kamal H.,Oh, Yi Seul,Fouad, Mohamed,Al-Jassir, Fawzi F.,Woo, Heung-Myong,Morsi, Yosry,Mo, Xiumei,Ivanovski, Saš,o Elsevier 2017 European polymer journal Vol.96 No.-

<P><B>Abstract</B></P> <P>Three dimensional (3D) constructs for vascular tissue engineering applications require scaffolds with highly porous architectures, high biocompatibility and mechanical stability. In this paper, composite fibrous tubular scaffolds composed of different ratios of poly(epsilon-caprolactone) (PCL) and polyamide-6 (PA-6) were simultaneously deposited layer by layer by employing the air jet spinning (AJS) textile technique. Specifically, we report on the optimal parameters for the fabrication of composite porous scaffolds that allow for precise control over the general scaffold architecture, as well as the physical and mechanical properties of the scaffolds. In vitro cell culture study was performed to investigate the influence of polymer composition and scaffold architecture on the adhesion of EA.hy926 human endothelial cells onto the fabricated scaffolds. The cell culture results indicated that a composite scaffold with low PA-6 fibrous content is the most promising substrate for EA.hy926 adhesion and proliferation. Based on the present findings, these highly porous composite tubular constructs support endothelial cell migration and cellular infiltration, and hence represent promising nano-fibrous scaffolds for vascular tissue engineering.</P> <P><B>Highlights</B></P> <P> <UL> <LI> PCL/Nylon 6 dual) fibrous 3D tissue scaffolds were synthesized for vascular grafts. </LI> <LI> Highly and tunable hybrid porous fibrous tissue scaffold was obtained by AJS. </LI> <LI> EA.hy926 EC was sued to determine the biocompatibility of tissue scaffolds. </LI> <LI> Dual scaffold provided a favorable attachment and proliferation of EA.hy926 human EC. </LI> <LI> Dual scaffold at low N6 content induced highest biocompatibility compared to others. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>