Fabrication of Polycaprolactone/Nano Hydroxyapatite (PCL/nHA) 3D Scaffold with Enhanced In Vitro Cell Response via Design for Additive Manufacturing (DfAM).

In this study, we investigated the dual-pore kagome-structure design of a 3D-printed scaffold with enhanced in vitro cell response and compared the mechanical properties with 3D-printed scaffolds with conventional or offset patterns. The compressive modulus of the 3D-printed scaffold with the proposed design was found to resemble that of the 3D-printed scaffold with a conventional pattern at similar pore sizes despite higher porosity. Furthermore, the compressive modulus of the proposed scaffold surpassed that of the 3D-printed scaffold with conventional and offset patterns at similar porosities owing to the structural characteristics of the kagome structure. Regarding the in vitro cell response, cell adhesion, cell growth, and ALP concentration of the proposed scaffold for 14 days was superior to those of the control group scaffolds. Consequently, we found that the mechanical properties and in vitro cell response of the 3D-printed scaffold could be improved by kagome and dual-pore structures through DfAM. Moreover, we revealed that the dual-pore structure is effective for the in vitro cell response compared to the structures possessing conventional and offset patterns.

Evaluation of the Antibacterial Activity and Cell Response for 3D-Printed Polycaprolactone/Nanohydroxyapatite Scaffold with Zinc Oxide Coating.

Among 3D-printed composite scaffolds for bone tissue engineering, researchers have been attracted to the use of zinc ions to improve the scaffold's anti-bacterial activity and prevent surgical site infection. In this study, we assumed that the concentration of zinc ions released from the scaffold will be correlated with the thickness of the zinc oxide coating on 3D-printed scaffolds. We investigated the adequate thickness of zinc oxide coating by comparing different scaffolds' characteristics, antibacterial activity, and in vitro cell response. The scaffolds' compressive modulus decreased as the zinc oxide coating thickness increased (10, 100 and 200 nm). However, the compressive modulus of scaffolds in this study were superior to those of other reported scaffolds because our scaffolds had a kagome structure and were made of composite material. In regard to the antibacterial activity and in vitro cell response, the in vitro cell proliferation on scaffolds with a zinc oxide coating was higher than that of the control scaffold. Moreover, the antibacterial activity of scaffolds with 100 or 200 nm-thick zinc oxide coating on Escherichia coli was superior to that of other scaffolds. Therefore, we concluded that the scaffold with a 100 nm-thick zinc oxide coating was the most appropriate scaffold to use as a bone-regenerating scaffold, given its mechanical property, its antibacterial activity, and its in vitro cell proliferation.

Evaluation of mechanical strength and bone regeneration ability of 3D printed kagome-structure scaffold using rabbit calvarial defect model.

In clinical conditions, the reconstructions performed in the complex and three-dimensional bone defects in the craniomaxillofacial (CMF) area are often limited in facial esthetics and jaw function. Furthermore, to regenerate a bone defect in the CMF area, the used scaffold should have unique features such as different mechanical strength or physical property suitable for complex shape and function of the CMF bones. Therefore, a three-dimensional synthetic scaffold with a patient-customized structure and mechanical properties is more suitable for the regeneration. In this study, the customized kagome-structure scaffold with complex morphology was assessed in vivo. The customized 3D kagome-structure model for the defect region was designed according to data using 3D computed tomography. The kagome-structure scaffold and the conventional grid-structure scaffold (as a control group) were fabricated using a 3D printer with a precision extruding deposition head using poly(ε-caprolactone) (PCL). The two types of 3D printed scaffolds were implanted in the 8-shaped defect model on the rabbit calvarium. To evaluate the osteoconductivity of the implanted scaffolds, new bone formation, hematoxylin and eosin staining, immunohistochemistry, and Masson's trichrome staining were evaluated for 16 weeks after implantation of the scaffolds. To assess the mechanical robustness and stability of the kagome-structure scaffold, numerical analysis considering the 'elastic-perfectly plastic' material properties and deformation under self-contact condition was performed by finite element analysis. As a result, the kagome-structure scaffold fabricated using 3D printing technology showed excellent mechanical robustness and enhanced osteoconductivity than the control group. Therefore, the 3D printed kagome-structure scaffold can be a better option for bone regeneration in complex and large defects than the conventional grid-type 3D printed scaffold.

Assessments for bone regeneration using the polycaprolactone SLUP (salt-leaching using powder) scaffold.

Salt-leaching using powder (SLUP) scaffolds are novel salt-leaching scaffolds with well-interconnected pores that do not require an organic solvent or high pressure. In this study, in vitro and in vivo cell behaviors were assessed using a PCL (polycaprolactone) SLUP scaffold. Moreover, using PCL, conventional salt-leaching and 3D-plotted scaffolds were fabricated as control scaffolds. Morphology, mechanical property, water absorption, and in vitro/in vivo cell response assessments were performed to clarify the characteristics of the SLUP scaffold compared with control scaffolds. Consequently, we verified that the interconnectivity between the pores of the SLUP scaffold was enhanced compared with conventional salt-leaching scaffolds. Moreover, in vitro cell attachment and proliferation of the SLUP scaffold were higher than those of the 3D-plotted scaffold because of their morphological characteristic. Furthermore, we revealed that new bone formation and bone ingrowth of the SLUP scaffold was superior to those of the calvarial defect model and 3D-plotted scaffold because of the high porosity and improved interconnectivity of pores by the SLUP technique without high pressure using powders. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3432-3444, 2017.

Mechanical properties and cell-culture characteristics of a polycaprolactone kagome-structure scaffold fabricated by a precision extruding deposition system.

To enhance the mechanical properties of three-dimensional (3D) scaffolds used for bone regeneration in tissue engineering, many researchers have studied their structure and chemistry. In the structural engineering field, the kagome structure has been known to have an excellent relative strength. In this study, to enhance the mechanical properties of a synthetic polymer scaffold used for tissue engineering, we applied the 3D kagome structure to a porous scaffold for bone regeneration. Prior to fabricating the biocompatible-polymer scaffold, the ideal kagome structure, which was manufactured by a 3D printer of the digital light processing type, was compared with a grid-structure, which was used as the control group, using a compressive experiment. A polycaprolactone (PCL) kagome-structure scaffold was successfully fabricated by additive manufacturing using a 3D printer with a precision extruding deposition head. To assess the physical characteristics of the fabricated PCL-kagome-structure scaffold, we analyzed its porosity, pore size, morphological structure, surface roughness, compressive stiffness, and mechanical bending properties. The results showed that, the mechanical properties of proposed kagome-structure scaffold were superior to those of a grid-structure scaffold. Moreover, Sarcoma osteogenic (Saos-2) cells were used to evaluate the characteristics of in vitro cell proliferation. We carried out cell counting kit-8 (CCK-8) and DNA contents assays. Consequently, the cell proliferation of the kagome-structure scaffold was increased; this could be because the surface roughness of the kagome-structure scaffold enhances initial cell attachment.

The fabrication of well-interconnected polycaprolactone/hydroxyapatite composite scaffolds, enhancing the exposure of hydroxyapatite using the wire-network molding technique.

In this study, the fabrication method was proposed for the well-interconnected polycaprolactone/hydroxyapatite composite scaffold with exposed hydroxyapatite using modified WNM technique. To characterize well-interconnected scaffolds in terms of hydroxyapatite exposure, several assessments were performed as follows: morphology, mechanical property, wettability, calcium ion release, and cell response assessments. The results of these assessments were compared with those of control scaffolds which were fabricated by precision extruding deposition (PED) apparatus. The control PED scaffolds have interconnected pores with nonexposed hydroxyapatite. Consequently, cell attachment of proposed WNM scaffold was improved by increased hydrophilicity and surface roughness of scaffold surface resulting from the exposure of hydroxyapatite particles and fabrication process using powders. Moreover, cell proliferation and differentiation of WNM scaffold were increased, because the exposure of hydroxyapatite particles may enhance cell adhesion and calcium ion release. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2315-2325, 2017.

Fabrication of dual-pore scaffolds using SLUP (salt leaching using powder) and WNM (wire-network molding) techniques.

In this study, a novel technique was proposed to fabricate dual-pore scaffolds combining both SLUP (salt leaching using powder) and WNM (wire-network molding) techniques. This technique has several advantages: solvent-free, no limit on the use of thermoplastic polymers as a raw material, and easiness of fabricating scaffolds with dual-scale pores that are interconnected randomized small pores. To fabricate dual-pore scaffolds, PCL and NaCl powders were mixed at a certain ratio. Subsequently, needles were inserted into a designed mold, and the mixture was filled into the mold thereafter. Subsequently, after the mold was pressurized, the mold was heated to melt the PCL powders. The PCL/NaCl structure and needles were separated from the mold. The structure was sonicated to leach-out the NaCl particles and was dried. Consequently, the remaining PCL structure became the dual-pore scaffold. To compare the characteristics of dual-pore scaffolds, control scaffolds, which are 3D plotter and SLUP scaffolds were fabricated.

Pre-S mutations of hepatitis B virus affect genome replication and expression of surface antigens.

BACKGROUNDS AND AIMS: In chronic hepatitis B virus (HBV) infection, quantitative HBV surface antigen (qHBsAg) is useful for monitoring viral replication and treatment responses. We aimed to determine whether pre-S mutations have any effect on circulating qHBsAg. METHODS: Plasmids expressing 1­8 amino acid deletion in pre-S1 ("pre-S1Δ1-8") and 3-25 amino acid deletion in pre-S2 ("pre-S2Δ3-25") were constructed. At 72 h posttransfection into Huh7 cells, qHBsAg were measured using electrochemiluminescence immunoassay analyzer. To mimic milieus of quasispecies, we co-transfected either pre-S1Δ1-8 or pre-S2Δ3-25 with wild type (WT). RESULTS: Pre-S mutations affected transcription and replication ability of HBV because of altered overlapping polymerase. Compared with WT, extracellular qHBsAg in pre-S1Δ1-8 and pre-S2Δ3-25 were on average 3.87-fold higher and 0.92-fold lower, respectively, whereas intracellular qHBsAg in pre-S1Δ1-8 and pre-S2Δ3-25 were 0.57-fold lower and 1.60-fold higher, respectively. Immunofluorescence staining of cellular HBsAg showed that pre-S1Δ1-8 had less staining and that pre-S2Δ3-25 had denser staining. As ratios of either pre-S1Δ1-8 or pre-S2Δ3-25:WT increased from 0:10 to 10:0 gradually, relative extracellular qHBsAg increased from 1.0 to 3.85 in pre-S1Δ1-8 co-transfection, whereas those decreased from 1.0 to 0.88 in pre-S2Δ3-25 co-transfection. CONCLUSION: Pre-S mutations exhibit different phenotypes of genome replication and HBsAg expression according to their locations. Thus, qHBsAg level for diagnosis and prognostification in chronic HBV infection should be used more cautiously, considering emergences of pre-S deletion mutants.