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Epidermal growth factor (EGF)-nanoparticle conjugates have the potential for cancer therapeutics due to the unique cytotoxic activity in cancer cells with EGF receptor (EGFR) overexpression. To gain its maximum activity, the EGF molecule should be immobilized on the nanoparticle surface in a defined orientation so as the bulky nanoparticle will not interfere EGF-EGFR interaction. Herein, we demonstrate successful enhancement of the anti-cancer activity of EGF-gold nanoparticle conjugates (EGF-GNPs) by controlling the EGF orientation on the surface of the nanoparticle through site-specific mutagenesis. Three lysine-free EGF variants (RR, RS, and SR) were designed, where two endogenous lysine residues were replaced with either arginine (R) or serine (S). The EGF mutants can be conjugated to the GNPs in a controlled orientation through the single amino group at the N-terminus. The ability of the mutants to induce extracellular signal-regulated kinase (ERK) phosphorylation was no different from wild type EGF (WT) in soluble form, rather lowered for one mutant (RR). However, after conjugated to GNPs, the SR mutants exhibited an enhanced biological activity than WT, in terms of ERK phosphorylation and growth inhibition of cancer cells. Further analysis of the binding constant of each mutant indicated the emergent enhanced activity of the GNP conjugates of the SR mutant was not solely contributed to the orientation, but to its higher binding activity to EGFR. These results validate the present genetic recombination strategy to improve the anticancer efficiency of EGF-GNPs., Graphical abstract

Photothermal therapy (PTT) has attracted broad attention as a promising method for cancer therapy with less severe side effects than conventional radiation therapy, chemotherapy and surgical resection. PTT relies on the photoconversion capacity of photothermal agents (PTAs), and a wide variety of nanomaterials have been employed as PTAs for cancer therapy due to their excellent photothermal properties. The PTAs are systematically or locally administered and become enriched in cancer cells to increase ablation efficiency. In recent years, PTAs and three-dimensional scaffolds have been hybridized to realize the local delivery of PTAs for the repeated ablation of cancer cells. Meanwhile, the composite scaffolds can stimulate the reconstruction and regeneration of the functional tissues and organs after ablation of cancer cells. A variety of composite scaffolds of photothermal nanomaterials have been prepared to combine the advantages of different modalities to maximize their therapeutic efficacy with minimal side effects. The synergistic effects make the composite scaffolds attractive for biomedical applications. This review summarizes these latest advances and discusses the future prospects., Graphical abstract

Bone apatite is not hydroxyapatite (HAp), it is carbonate apatite (CO3Ap), which contains 6–9 mass% carbonate in an apatitic structure. The CO3Ap block cannot be fabricated by sintering because of its thermal decomposition at the sintering temperature. Chemically pure (100%) CO3Ap artificial bone was recently fabricated through a dissolution–precipitation reaction in an aqueous solution using a precursor, such as a calcium carbonate block. In this paper, methods of fabricating CO3Ap artificial bone are reviewed along with their clinical and animal results. CO3Ap artificial bone is resorbed by osteoclasts and upregulates the differentiation of osteoblasts. As a result, CO3Ap demonstrates much higher osteoconductivity than HAp and is replaced by new bone via bone remodeling. Granular-type CO3Ap artificial bone was approved for clinical use in Japan in 2017. Honeycomb-type CO3Ap artificial bone is fabricated using an extruder and a CaCO3 honeycomb block as a precursor. Honeycomb CO3Ap artificial bone allows vertical bone augmentation. A CO3Ap-coated titanium plate has also been fabricated using a CaCO3-coated titanium plate as a precursor. The adhesive strength is as high as 76.8 MPa, with excellent tissue response and high osteoconductivity., Graphical abstract

To improve the therapeutic potential of β-cyclodextrin (β-CD)-threaded acid-degradable polyrotaxanes (β-CD PRXs) in cholesterol-related metabolic disorders, we investigated the effect of carboxylation of β-CD PRXs on intracellular uptake. In this study, we established a synthetic method for the modification of carboxylalkyl carbamates on β-CD PRXs without degradation and synthesized three series of carboxyalkyl carbamate group-modified β-CD PRXs with different alkyl spacer lengths. The modification of carboxymethyl carbamate (CMC), carboxyethyl carbamate (CEC), and carboxypropyl carbamate (CPC) on the β-CD PRXs slightly reduced the interaction of the PRXs with the lipid layer model compared with the modification of 2-(2-hydroxyethoxy)ethyl carbamate (HEE-PRX), which was used in our previous studies. However, all the carboxylated β-CD PRXs showed a significantly stronger interaction with a protein model compared with HEE-PRX. The carboxylated β-CD PRXs showed significantly high intracellular uptake, through macrophage scavenger receptor A (MSR-A)-mediated endocytosis, in MSR-A-positive RAW 264.7 cells compared with HEE-PRX. Interestingly, the carboxylated β-CD PRXs also showed significantly higher intracellular uptake even in MSR-A-negative cells compared with HEE-PRX. Carboxylated β-CD PRXs are considered to strongly interact with other membrane proteins, resulting in high intracellular uptake. The length of the alkyl spacer affected the intracellular uptake levels of carboxylated PRXs, however, this relationship was varied for different cell types. Furthermore, none of the carboxylated β-CD PRXs exhibited cytotoxicity in the RAW 264.7 and NIH/3T3 cells. Altogether, carboxylation of β-CD PRXs is a promising chemical modification approach for their therapeutic application because carboxylated β-CD PRXs exhibit high cellular internalization efficiency in MSR-A-negative cells and negligible toxicity., GRAPHICAL ABSTRACT

Much research has been conducted on fabricating biomimetic biomaterials in vitro. Tissue engineering approaches are often conducted by combining cells, scaffolds, and growth factors. However, the degradation rate of scaffolds is difficult to control and the degradation byproducts occasionally limit tissue regeneration. To overcome these issues, we have developed a novel system using a thermo-responsive hydrogel that forms scaffold-free, three-dimensional (3D) cell constructs with arbitrary size and morphology. 3D cell constructs prepared using bone marrow-derived stromal stem cells (BMSCs) exhibited self-organizing ability and formed bone-like tissue with endochondral ossification. Endothelial cells were then introduced into the BMSC construct and a vessel-like structure was formed within the constructs. Additionally, the bone formation ability was promoted by endothelial cells and cell constructs could be freeze-dried to improve their clinical application. A pre-treatment with specific protein protectant allowed for the fabrication of novel bone substitutes composed only of cells. This 3D cell construct technology using thermo-responsive hydrogels was then applied to other cell species. Cell constructs composed of dental pulp stem cells were fabricated, and the resulting construct regenerated pulp-like tissue within a human pulpless tooth. In this review, we demonstrate the approaches for the in vitro fabrication of bone and dental pulp-like tissue using thermo-responsive hydrogels and their potential applications., Graphical abstract

In this study, we designed and synthesized an implantable anti-CD25 antibody-immobilized polyethylene (CD25-PE) mesh to suppress tumor growth by removing regulatory T cells (Tregs). The PE mesh was graft-polymerized with poly(acrylic acid), and the anti-mouse CD25 antibody was then immobilized using the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide reaction. Immobilization of the antibody on the PE mesh was confirmed by immunostaining. The CD25-PE mesh could effectively and selectively capture CD25-positive cells through antigen-antibody interactions when the CD25-PE mesh was incubated with a suspension of mouse spleen cells, including CD25-positive cells. In addition, implantation of the CD25-PE mesh into mice subcutaneously demonstrated the Treg-capturing ability of the CD25-PE mesh with only a weak inflammatory reaction. In tumor-bearing mice, tumor growth was suppressed by subcutaneous implantation of the CD25-PE mesh near the tumor for 1 week. These results suggested that the anti-CD25 antibody-immobilized material could capture Tregs in vivo and inhibit tumor proliferation in a limited tumor-bearing mouse model. Further research is needed to facilitate cancer immunotherapy using implantable anti-CD25 antibody-immobilized material as a Treg-capturing device., Graphical abstract

The growing prevalence of vascular diseases worldwide has emphasized the need for novel tissue-engineered options concerning the development of vascularized 3D constructs. This study reports, for the first time, the use of external magnetic fields to stimulate mesenchymal stromal cells (MSCs) to increase the production of vascular endothelial growth factor-A (VEGF-A). Polyvinylalcohol and gelatin-based scaffolds, containing iron oxide nanoparticles, were designed for optimal cell magnetic stimulation. While the application of static magnetic fields over 24 h did not impact on MSCs proliferation, viability and phenotypic identity, it significantly increased the production of VEGF-A and guided MSCs morphology and alignment. The ability to enhance MSCs angiogenic potential was demonstrated by the increase in the number of new vessels formed in the presence of MSCs conditioned media through in vitro and in vivo models. Ultimately, this study uncovers the potential to manipulate cellular processes through short-term magnetic stimulation., Graphical abstract

In recent years remarkable efforts have been made to produce artificial bone through tissue engineering techniques. Silk fibroin (SF) and hydroxyapatite (HA) have been used in bone tissue regeneration as biomaterials due to mechanical properties of SF and biocompatibility of HA. There has been growing interest in developing SF/HA composites to reduce bone defects. In this regard, several attempts have been made to study the biocompatibility and osteoconductive properties of this material. This article overviews the recent advance from last few decades in terms of the preparative methods and application of SF/HA in bone regeneration. Its first part is related to SF that presents the most common sources, preparation methods and comparison of SF with other biomaterials. The second part illustrates the importance of HA by providing information about its production and properties. The third part presents comparative studies of SF/HA composites with different concentrations of HA along with methods of preparation of composites and their applications., Graphical abstract

This study proposes to develop a dual-acting antibacterial film of porous chitosan (Cs) embedded with small molecular compound, which possesses photosensitive characteristics with bactericidal efficacy, to promote the accelerated recovery of infectious wounds. The Cs/small molecular compound (Cs-cpd.2) dressing was prepared using the freeze-drying method. Characterization of the synthesized Cs-cpd.2 indicated that it has high porosity and moisture absorption effect, hence enhancing the absorption of wound exudate. Experimental results showed that Cs-cpd.2 dressing has good bactericidal and bacteriostatic effects on Staphylococcus aureus under visible-light irradiation and has antibacterial effect in the dark. It was also found that the small molecular compound does not have cytotoxicity at a dose of 0–5 μM. Furthermore, Cs-cpd.2 that contained small molecular compound with a concentration of 0.3–1 μM has positive effect on both the cell viability rate and cell proliferation rate of human fibroblast CG1639. Cs-cpd.2 can significantly promote cell proliferation when the small molecular compound and the basic fibroblast growth factor bFGF were added together. Therefore, the proposed Cs-cpd.2 dressing is feasible for photodynamic therapy (PDT) and clinical wound dressing applications., GRAPHICAL ABSTRACT

In mechanotransduction studies, flow-induced shear stress (FSS) is often applied to two-dimensional (2D) cultured cells with a parallel-plate flow chamber (PPFC) due to its simple FSS estimation. However, cells behave differently under FSS inside a 3D scaffold (e.g. 10 mPa FSS was shown to induce osteogenesis of human mesenchymal stem cells (hMSC) in 3D but over 900 mPa was needed for 2D culture). Here, a simple in vitro biomimetic perfusion system using borosilicate glass capillary tubes has been developed to study the cellular behaviour under low-level FSS that mimics 3D culture. It has been shown that, compared to cells in the PPFC, hMSC in the capillary tubes had upregulated Runx-2 expression and osteogenic cytoskeleton actin network under 10 mPa FSS for 24 h. Also, an image analysis method based on Haralick texture measurement has been used to identify osteogenic actin network. The biomimetic perfusion system can be a valuable tool to study mechanotransduction in 3D for more clinical relevant tissue-engineering applications., Graphical abstract

A variety of poly(N-isopropylacrylamide) (PIPAAm)-grafted surfaces have been reported for temperature-controlled cell adhesion/detachment. However, the surfaces reported to date need further improvement to achieve good outcomes for both cell adhesion and detachment, which are inherently contradictory behaviors. This study investigated the effects of terminal cationization and length of grafted PIPAAm chains on temperature-dependent cell behavior. PIPAAm brushes with three chain lengths were constructed on glass coverslips via surface-initiated reversible addition-fragmentation chain transfer (RAFT) polymerization. Terminal substitution of the grafted PIPAAm chains with either monocationic trimethylammonium or nonionic isopropyl moieties was performed through the reduction of terminal RAFT-related groups and subsequent thiol-ene reaction with the corresponding acrylamide derivatives. Although the thermoresponsive properties of the PIPAAm brush surfaces were scarcely affected by the terminal functional moiety, the zeta potentials of the cationized PIPAAm surfaces were higher than those of the nonionized ones, both below and above the phase transition temperature of PIPAAm (30°C). When bovine endothelial cells were cultured on each surface at 37°C, the number of adherent cells decreased with longer PIPAAm. Notably, cell adhesion on the cationized PIPAAm surfaces was higher than that on the nonionized surfaces. This terminal effect on cell adhesion gradually weakened with increasing PIPAAm length. In particular, long-chain PIPAAm brushes virtually showed cell repellency even at 37°C, regardless of the termini. Interestingly, moderately long-chain PIPAAm brushes promoted cell detachment at 20°C, with negligible terminal electrostatic interruption. Consequently, both cell adhesion and detachment were successfully improved by choosing an appropriate PIPAAm length with terminal cationization., Graphical abstract

The microvascular network is a simple but critical system that is responsible for a range of important biological mechanisms in the bodies of all animals. The ability to generate a functional microvessel not only makes it possible to engineer vital tissue of considerable size but also serves as a platform for biomedical studies. However, most of the current methods for generating microvessel networks in vitro use rectangular channels which cannot represent real vessels in vivo and have dead zones at their corners, hence hindering the circulation of culture medium. We propose a scaffold-wrapping method which enables fabrication of a customized microvascular network in vitro in a more biomimetic way. By integrating microelectromechanical techniques with thermal reflow, we designed and fabricated a microscale hemi-cylindrical photoresist template. A replica mold of polydimethylsiloxane, produced by casting, was then used to generate cylindrical scaffolds with biodegradable poly(lactide-co-glycolide) (PLGA). Human umbilical vein endothelial cells were seeded on both sides of the PLGA scaffold and cultured using a traditional approach. The expression of endothelial cell marker CD31 and intercellular junction vascular endothelial cadherin on the cultured cell demonstrated the potential of generating a microvascular network with a degradable cylindrical scaffold. Our method allows cells to be cultured on a scaffold using a conventional culture approach and monitors cell conditions continuously. We hope our cell-covered scaffold can serve as a framework for building large tissues or can be used as the core of a vascular chip for in vitro circulation studies.

Constructing satisfied small-diameter vascular graft (diameter less than 6 mm) remains an unsolvable challenge in vascular tissue engineering. This study described the fabrication of electrospun polyurethane/polycaprolactone (PU/PCL) membranes chemically grafted with various densities of conjugated linoleic acid (CLA) – an antithrombotic fatty acid – for making small-diameter blood vessel. Differences in mechanical, antithrombotic properties and biocompatibility of the membranes resulting from the CLA-grafting procedure were the focus of the study. Investigation of mechanical properties relevant to vascular graft application revealed that these properties of the membranes remained unaffected and satisfied clinical criteria following the CLA graft. Blood–membrane interaction assays showed that the CLA-grafted membranes mitigated the adhesion of blood cells, as well as preventing blood coagulation. These effects were also commensurate with increasing density of CLA, suggesting an effective approach to improve antithromboticity. Cellular tests suggested that CLA has an optimal density at which it promoted cell proliferation on the surface of the membranes; however, excessive presence of CLA might cause undesirable inhibition on cells. In conclusion, PU/PCL membrane grafted with CLA could be a prospective material for vascular tissue engineering with further development and investigation., Graphical Abstract

Since circulating tumor cells (CTCs) are tumor cells which are found in the blood of cancer patients, CTCs are potential tumor markers, so a rapid isolation of CTCs is desirable for clinical applications. In this paper, a three-dimensional polystyrene (PS) microfiber fabric with vacuum aspiration system was developed for capturing CTCs within a short time. Various microfiber fabrics with different diameters were prepared by the electrospinning method and optimized for contact frequency with cells. Vacuum aspiration utilizing these microfiber fabrics could filter all cells within seconds without mechanical damage. The microfiber fabric with immobilized anti-EpCAM antibodies was able to specifically capture MCF-7 cells that express EpCAM on their surfaces. The specificity of the system was confirmed by monitoring the ability to isolate MCF-7 cells from a mixture containing CCRF-CEM cells that do not express EpCAM. Furthermore, the selective capture ability of the microfiber was retained even when the microfiber was exposed to the whole blood of pigs spiked with MCF-7 cells. The specific cell capture ratio of the vacuum aspiration system utilizing microfiber fabric could be improved by increasing the thickness of the microfiber fabric through electrospinning time.