Fiber-reinforced scaffolds in soft tissue engineering.

Soft tissue engineering has been developed as a new strategy for repairing damaged or diseased soft tissues and organs to overcome the limitations of current therapies. Since most of soft tissues in the human body are usually supported by collagen fibers to form a three-dimensional microstructure, fiber-reinforced scaffolds have the advantage to mimic the structure, mechanical and biological environment of natural soft tissues, which benefits for their regeneration and remodeling. This article reviews and discusses the latest research advances on design and manufacture of novel fiber-reinforced scaffolds for soft tissue repair and how fiber addition affects their structural characteristics, mechanical strength and biological activities in vitro and in vivo. In general, the concept of fiber-reinforced scaffolds with adjustable microstructures, mechanical properties and degradation rates can provide an effective platform and promising method for developing satisfactory biomechanically functional implantations for soft tissue engineering or regenerative medicine.

A novel method to in vitro evaluate biocompatibility of nanoscaled scaffolds.

This study provided a new method to in vitro evaluate the biocompatibility of nanoscaled scaffolds for tissue engineering with neutrophils other than ordinary cell culture. The neutrophils were separated from human peripheral blood of healthy subjects. In vitro degradation product of nanohydroxyapatite/collagen (nHAC), nanohydroxyapatite/collagen/poly (L-lactic acid) (nHACP), and nHACP reinforced by chitin fibers (nHACP/CF) in the D-Hank's Balanced Salt Solution (D-HBSS) was used as the testing solution, which was thereafter mixed with the neutrophils. It was shown that the cell survival rate in the testing solutions had no significant difference from that in the D-HBSS (control). However, from both gene and protein expression levels, the lactate dehydrogenase and tumor necrosis factor-alpha of the neutrophils in the nHACP/CF testing solution were found lowest during the whole testing period; the main reasons of which might be that the calcium release rate of the scaffold was slowest and that the pH value of its degradation solution was nearest to that of human body. Moreover, in vivo experiments showed that most inflammation reactions happened for nHAC and poly (L-lactic acid) groups, while the least inflammation reactions happened for nHACP/CF group in the subcutaneous dorsum of mice at 2 weeks after the surgery, which confirmed the in vitro findings. These results indicated that the pH value and the certain metal iron concentration of the nanoscaled scaffold degradation solution should be two important factors that significantly affect its biocompatibility. This study provides a simple and effective biocompatibility test method for biodegradable nanoscaled tissue engineering scaffolds. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2117-2125, 2016.

Current investigations into magnetic nanoparticles for biomedical applications.

It is generally recognized that nanoparticles possess unique physicochemical properties that are largely different from those of conventional materials, specifically the electromagnetic properties of magnetic nanoparticles (MNPs). These properties have attracted many researchers to launch investigations into their potential biomedical applications, which have been reviewed in this article. First, common types of MNPs were briefly introduced. Then, the biomedical applications of MNPs were reviewed in seven parts: magnetic resonance imaging (MRI), cancer therapy, the delivery of drugs and genes, bone and dental repair, tissue engineering, biosensors, and in other aspects, which indicated that MNPs possess great potentials for many kinds of biomedical applications due to their unique properties. Although lots of achievements have been obtained, there is still a lot of work to do. New synthesis techniques and methods are still needed to develop the MNPs with satisfactory biocompatibility. More effective methods need to be exploited to prepare MNPs-based composites with fine microstructures and high biomedical performances. Other promising research points include the development of more appropriate techniques of experiments both in vitro and in vivo to detect and analyze the biocompatibility and cytotoxicity of MNPs and understand the possible influencing mechanism of the two properties. More comprehensive investigations into the diagnostic and therapeutic applications of composites containing MNPs with "core-shell" structure and deeper understanding and further study into the properties of MNPs to reveal their new biomedical applications, are also described in the conclusion and perspectives part.

The applications of conductive nanomaterials in the biomedical field.

As their name suggests, conductive nanomaterials (CNMs) are a type of functional materials, which not only have a high surface area to volume ratio, but also possess excellent conductivity. Thus far, CNMs have been widely used in biomedical applications, such as effectively transferring electrical signals, and providing a large surface area to adsorb proteins and induce cellular functions. Recent works propose further applications of CNMs in biosensors, tissue engineering, neural probes, and drug delivery. This review focuses on common types of CNMs and elaborates on their unique properties, which indicate that such CNMs have a potential to develop into a class of indispensable biomaterials for the diagnosis and therapy of human diseases.

Effects of physicochemical properties of nanomaterials on their toxicity.

Due to their unique size and properties, nanomaterials have numerous applications, which range from electronics, cosmetics, household appliances, energy storage, and semiconductor devices, to medical products such as biological sensors, drug carriers, bioprobes, and implants. Many of the promising properties of nanomaterials arise from their large surface to volume ratio and, therefore, nanobiomaterials that are implantable have a large contact area with the human body. Before, therefore, we can fully exploit nanomaterials, in medicine and bioengineering; it is necessary to understand how they can affect the human body. As a step in this direction, this review paper provides a comprehensive summary of the effects that the physicochemical properties of commonly used nanobiomaterials have on their toxicity. Furthermore, the possible mechanisms of toxicity are described with the aim to provide guidance concerning the design of the nanobiomaterials with desirable properties.

Suppression of c-Myc is involved in multi-walled carbon nanotubes' down-regulation of ATP-binding cassette transporters in human colon adenocarcinoma cells.

Over-expression of ATP-binding cassette (ABC) transporters, a large family of integral membrane proteins that decrease cellular drug uptake and accumulation by active extrusion, is one of the major causes of cancer multi-drug resistance (MDR) that frequently leads to failure of chemotherapy. Carbon nanotubes (CNTs)-based drug delivery devices hold great promise in enhancing the efficacy of cancer chemotherapy. However, CNTs' effects on the ABC transporters remain under-investigated. In this study, we found that multiwalled carbon nanotubes (MWCNTs) reduced transport activity and expression of ABC transporters including ABCB1/Pgp and ABCC4/MRP4 in human colon adenocarcinoma Caco-2 cells. Proto-oncogene c-Myc, which directly regulates ABC gene expression, was concurrently decreased in MWCNT-treated cells and forced over-expression of c-Myc reversed MWCNTs' inhibitory effects on ABCB1 and ABCC4 expression. MWCNT-cell membrane interaction and cell membrane oxidative damage were observed. However, antioxidants such as vitamin C, ß-mecaptoethanol and dimethylthiourea failed to antagonize MWCNTs' down-regulation of ABC transporters. These data suggest that MWCNTs may act on c-Myc, but not through oxidative stress, to down-regulate ABC transporter expression. Our findings thus shed light on CNTs' novel cellular effects that may be utilized to develop CNTs-based drug delivery devices to overcome ABC transporter-mediated cancer chemoresistance.

DNA/Ag Nanoparticles as Antibacterial Agents against Gram-Negative Bacteria.

Silver (Ag) nanoparticles were produced using DNA extracted from salmon milt as templates. Particles spherical in shape with an average diameter smaller than 10 nm were obtained. The nanoparticles consisted of Ag as the core with an outermost thin layer of DNA. The DNA/Ag hybrid nanoparticles were immobilized over the surface of cotton based fabrics and their antibacterial efficiency was evaluated using E. coli as the typical Gram-negative bacteria. The antibacterial experiments were performed according to the Antibacterial Standard of Japanese Association for the Functional Evaluation of Textiles. The fabrics modified with DNA/Ag nanoparticles showed a high enough inhibitory and killing efficiency against E. coli at a concentration of Ag ≥ 10 ppm.

Resin composites reinforced by nanoscaled fibers or tubes for dental regeneration.

It has been stated clearly that nanofillers could make an enhancement on the mechanical performances of dental composites. In order to address current shortage of traditional dental composites, fillers in forms of nanofibers or nanotubes are broadly regarded as ideal candidates to greatly increase mechanical performances of dental composites with low content of fillers. In this review, the efforts using nanofibers and nanotubes to reinforce mechanical performances of dental composites, including polymeric nanofibers, metallic nanofibers or nanotubes, and inorganic nanofibers or nanotubes, as well as their researches related, are demonstrated in sequence. The first purpose of current paper was to confirm the enhancement of nanofibers or nanotubes' reinforcement on the mechanical performances of dental restorative composite. The second purpose was to make a general description about the reinforcement mechanism of nanofibers and nanotubes, especially, the impact of formation of interphase boundary interaction and nanofibers themselves on the advanced mechanical behaviors of the dental composites. By means of the formation of interface interaction and poststretching nanofibers, reinforced effect of dental composites by sorts of nanofibers/nanotubes has been successfully obtained.

Bone enhancing effect of titanium-binding proteins isolated from bovine bone and implanted into rat calvaria with titanium scaffold.

Based on our previous finding that a chromatography with titanium beads selectively binds phosphoproteins, including caseins, phosvitin and dentin phosphoproteins, we investigated whether bone phosphoproteins also bind to titanium. Bovine bone matrix proteins were extracted with 2 M urea/PBS after demineralization. The 2 M urea extract was directly applied to the titanium chromatography column as reported. The chromatogram showed an initial large peak at breakthrough position (non-binding fraction) and a smaller second peak eluted later (titanium-binding fraction). Both peaks were analyzed by SDS polyacrylamide gel electrophoresis. Stains-all staining which preferentially identifies phospho-proteins revealed that the first peak contained no positively stained band, while the second peak showed 4 or 5 distinctive bands indicative of bone phosphoproteins. To investigate the biological functions of the titanium-binding bone proteins (TiBP), we implanted them into calvaria of rats, combined with titanium web (TW), a highly porous titanium scaffold of thin titanium-fibers. Bone TiBP induced significantly enhanced bone formation, and new bone appeared connected directly to titanium fibers, accompanied by active blood vessel formations. Control TW alone did not induce bone formation within the titanium framework. These results demonstrate that the bone titanium-binding proteins include phosphoproteins which enhance bone formation when implanted into bone with titanium.

Photophysical properties and biocompatibility of Photoluminescent Y2O3:Eu nanoparticles in polymethylmetacrylate matrix.

In this study, we produced europium-doped yttoria (Y2O3:Eu) nanoparticles and investigated their photoluminescent properties and biocompatibility. The Y2O3:Eu nanoparticles showed excellent photoluminescent properties and cytocompatibility. We also analyzed the photophysical properties of the nanoparticles in PMMA films. When the Y2O3:Eu nanoparticles were incorporated in the polymer film, they showed a strong red emission spectrum, similar to that seen with the particles alone. Energy dispersive X-ray spectroscopy (EDS) measurements indicated that the particles were distributed homogeneously in the PMMA film. Such materials could be applied not only to optoelectronic devices but also to biomedical applications such as bioimaging tools or luminescent medical/dental adhesive materials.

Polyglycerol-coated nanodiamond as a macrophage-evading platform for selective drug delivery in cancer cells.

A successful targeted drug delivery device for cancer chemotherapy should ideally be able to avoid non-specific uptake by nonmalignant cells, particularly the scavenging monocyte-macrophage system as well as targeting efficacy to bring the drug preferentially into tumor cells. To this purpose, we developed a platform based on detonation nanodiamond (dND) with hyperbranched polyglycerol (PG) coating (dND-PG). dND-PG was first demonstrated to evade non-specific cell uptake, particularly by macrophages (U937). RGD targeting peptide was then conjugated to dND-PG through multistep organic transformations to yield dND-PG-RGD that still evaded macrophage uptake but was preferentially taken up by targeted A549 cancer cells (expressing RGD peptide receptors). dND-PG and dND-PG-RGD showed good aqueous solubility and cytocompatibitlity. Subsequently, the anticancer agent doxorubicin (DOX) was loaded through acid-labile hydrazone linkage to yield dND-PG-DOX and dND-PG-RGD-DOX. Their cellular uptake and cytotoxicity were compared against DOX in A549 cells and U937 macrophages. It was found that dND-PG-DOX uptake was substantially reduced, displaying little toxicity in either type of cells by virtue of PG coating, whereas dND-PG-RGD-DOX exerted selective toxicity to A549 cells over U937 macrophages that are otherwise highly sensitive to DOX. Finally, dND-PG was demonstrated to have little influence on U937 macrophage cell functions, except for a slight increase of TNF-α production in resting U937 macrophages. dND-PG is a promising drug carrier for realization of highly selective drug delivery in tumor cells through specific uptake mechanisms, with minimum uptake in and influence on macrophages.

Biocomposites reinforced by fibers or tubes as scaffolds for tissue engineering or regenerative medicine.

As a dynamic and hierarchically organized composite, native extracellular matrix (ECM) not only supplies mechanical support, which the embedded cells need, but also regulates various cellular activities through interaction with them. On the basis of the ECM-mimetic principle, good biocompatibility and appropriate mechanical properties are the two basic requirements that the ideal scaffolds for the tissue engineering or regenerative medicine need. Some fibers and tubes have been shown effective to reinforce scaffolds for tissue engineering or regenerative medicine. In this review, three parts, namely properties affected by the addition of fibers or tubes, scaffolds reinforced by fibers or tubes for soft tissue repair, and scaffolds reinforced by fibers or tubes for hard tissue repair are stated, which shows that tissue repair or regeneration efficacy was enhanced significantly by fiber or tube reinforcement. In addition, it indicates that these reinforcing agents can improve the biocompatibility and biodegradation of the scaffolds in most cases. However, there are still some concerns, such as the homogeneousness in structure or composition throughout the reinforced scaffolds, the adhesive strength between the matrix and the fibers or tubes, cytotoxicity of nanoscaled reinforcing agents, etc., which were also discussed in the conclusion and perspectives part.

Carbon nanotubes functionalized with fibroblast growth factor accelerate proliferation of bone marrow-derived stromal cells and bone formation.

Multi-walled carbon nanotubes (MWCNTs) were functionalized with fibroblast growth factor (FGF) and the advantages of their use as scaffolds for bone augmentation were evaluated in vitro and in vivo. The activity of FGF was assessed by measuring the effect on the proliferation of rat bone marrow stromal cells (RBMSCs). The presence of FGF enhanced the proliferation of RBMSCs and the FGF covalently conjugated to the nanotubes (FGF-CNT) showed the same effect as FGF alone. In addition, FGF-CNT coated sponges were implanted between the parietal bone and the periosteum of rats and the formation of new bone was investigated. At day 14 after implantation, a larger amount of newly formed bone was clearly observed in most pores of FGF-CNT coated sponges. These findings indicated that MWCNTs accelerated new bone formation in response to FGF, as well as the integration of particles into new bone during its formation. Scaffolds coated with FGF-CNT could be considered as promising novel substituting materials for bone regeneration in future tissue engineering applications.

An in vitro evaluation of the biological effects of carbon nanotube-coated dental zirconia.

THE PURPOSE OF THIS STUDY IS TO EVALUATE FUNCTIONALIZED MULTIWALLED CARBON NANOTUBES (FMWCNTS) AS A POTENTIAL COATING MATERIAL FOR DENTAL ZIRCONIA FROM A BIOLOGICAL PERSPECTIVE: its effect on cell proliferation, viability, morphology, and the attachment of an osteoblast-like cell. Osteoblast-like (Saos-2) cells were seeded on uncoated and fMWCNT-coated zirconia discs and in culture dishes that served as controls. The seeding density was 10(4) cells/cm(2), and the cells were cultured for 6 days. Cell viability, proliferation and attachment of the Saos-2 cells were studied. The results showed that Saos-2 cells were well attached to both the uncoated and the fMWCNT-coated zirconia discs. Cell viability and proliferation on the fMWCNT-coated zirconia discs were almost the same as for the control discs. Better cell attachment was seen on the fMWCNT-coated than on the uncoated zirconia discs. In conclusion, fMWCNTs seem to be a promising coating material for zirconia-based ceramic surfaces to increase the roughness and thereby enhance the osseointegration of zirconia implants.

Long-term biopersistence of tangled oxidized carbon nanotubes inside and outside macrophages in rat subcutaneous tissue.

Because of their mechanical strength, chemical stability, and low molecular weight, carbon nanotubes (CNTs) are attractive biological implant materials. Biomaterials are typically implanted into subcutaneous tissue or bone; however, the long-term biopersistence of CNTs in these tissues is unknown. Here, tangled oxidized multi-walled CNTs (t-ox-MWCNTs) were implanted into rat subcutaneous tissues and structural changes in the t-ox-MWCNTs located inside and outside of macrophages were studied for 2 years post-implantation. The majority of the large agglomerates were present in the intercellular space, maintained a layered structure, and did not undergo degradation. By contrast, small agglomerates were found inside macrophages, where they were gradually degraded in lysosomes. None of the rats displayed symptoms of cancer or severe inflammatory reactions such as necrosis. These results indicate that t-ox-MWCNTs have high biopersistence and do not evoke adverse events in rat subcutaneous tissue in vivo, demonstrating their potential utility as implantable biomaterials.

Nanostructured scaffolds for bone tissue engineering.

It has been demonstrated that nanostructured materials, compared with conventional materials, may promote greater amounts of specific protein interactions, thereby more efficiently stimulating new bone formation. It has also been indicated that, when features or ingredients of scaffolds are nanoscaled, a variety of interactions can be stimulated at the cellular level. Some of those interactions induce favorable cellular functions while others may leads to toxicity. This review presents the mechanism of interactions between nanoscaled materials and cells and focuses on the current research status of nanostructured scaffolds for bone tissue engineering. Firstly, the main requirements for bone tissue engineering scaffolds were discussed. Then, the mechanism by which nanoscaled materials promote new bone formation was explained, following which the current research status of main types of nanostructured scaffolds for bone tissue engineering was reviewed and discussed.

Interaction between titanium and phosphoproteins revealed by chromatography column packed with titanium beads.

The biochemical mechanism behind the strong binding between titanium and living bone has not been fully elucidated, in spite of worldwide clinical application of this phenomenon. We hypothesized that one of the core mechanisms may reside in the interaction between certain proteins in the host tissues and the implanted titanium. To verify the interaction between titanium and proteins, we chose the technique of chromatography in that titanium spherical beads (45 µm) were packed into a column to obtain a bed volume of 16×50 mm, which was eluted with phosphate buffered saline (PBS) and a straight gradient system made by using PBS and 25 mM NaOH. Fetal calf serum, albumin, lysozyme, casein, phosvitin and dentin phosphoprotein (phosphophoryn) were applied to the column. Most part of albumin and lysozyme eluted with the breakthrough peak, indicating practically no affinity to titanium. Fetal bovine serum also eluted mostly as the breakthrough peak, but distinct retained peak was observed. On the other hand, α-casein, phosvitin and phosphophoryn exhibited a distinct retained peak separated from the breakthrough peak. We proposed that phosphate groups (phosphoserines) in the major phosphoproteins, α-casein, phosvitin and phosphophoryn may be involved in the binding of these proteins with titanium.

Biodistribution of aqueous suspensions of carbon nanotubes in mice and their biocompatibility.

In this study, we prepared two-types of water-dispersible carbon nanotubes (CNTs) and investigated their biodistribution in mice as well as bio-/cyto-compatibility. After administration, their organs were excised at various post-injection times, then observed using both optical and transmission electron microscopy (TEM). The color of the liver and lung markedly darkened, suggesting that administered CNTs reached these organs. By TEM observation, the CNTs were found in the liver and lung. They were observed even in the kidney and spleen, though their distributions in those organs were very low compared with that in liver and lung. Therefore, most of the administered CNTs would be accumulated in the liver or lung. However, the time profile of the body weight of CNT-administered mice was close to that of control mice. In addition, we estimated the cytocompatibility of the water-dispersible CNTs for hepatocytes. According to a TNF-alpha assay of the cells cultured with CNTs, the expression level was almost the same as that of the control. These results suggested that the water-dispersible CNTs have good bio-/cyto-compatibility under this condition.

The use of carbon nanotubes to induce osteogenic differentiation of human adipose-derived MSCs in vitro and ectopic bone formation in vivo.

Carbon nanotubes (CNTs), one of the most concerned nanomaterials, with unique electrical, mechanical and surface properties, have been shown suitable for biomedical application. In this study, we evaluated attachment, proliferation, osteogenic gene expression, ALP/DNA, protein/DNA and mineralization of human adipose-derived stem cells cultured in vitro on multi-walled carbon nanotubes (MWNTs) and graphite (GP) compacts with the same dimension. Moreover, we assessed the effect of these two kinds of compacts on ectopic bone formation in vivo. First of all, higher ability of the MWNTs compacts to adsorb proteins, comparing with the GP compacts, was shown. During the conventional culture, it was shown that MWNTs could induce the expression of ALP, cbfa1 and COLIA1 genes while GP could not. Furthermore, alkaline phosphatase (ALP)/DNA and protein/DNA of the cell on the MWNTs compacts, was significantly higher than those of the cells on the GP compacts. With the adsorption of the proteins in culture medium with 50% fetal bovine serum (FBS) in advance, the increments of the ALP/DNA and protein/DNA for the MWNTs compacts were found respectively significantly more than the increments of those for the GP compacts, suggesting that the larger amount of protein adsorbed on the MWNTs was crucial. More results showed that ALP/DNA and protein/DNA of the cells on the two kinds of compacts pre-soaked in culture medium having additional rhBMP-2 were both higher than those of the cells on the samples re-soaked in culture medium with 50% FBS, and that those values for the MWNTs compacts increased much more. Larger mineral content was found on the MWNTs compacts than on the GP compacts at day 7. In vivo experiment showed that the MWNTs could induce ectopic bone formation in the dorsal musculature of ddy mice while GP could not. The results indicated that MWNTs might stimulate inducible cells in soft tissues to form inductive bone by concentrating more proteins, including bone-inducing proteins.

Phase transformation of zirconia ceramics by hydrothermal degradation.

Zirconia has found wide application in dentistry because of its high mechanical strength and superior esthetic properties. However, zirconia degradation caused by phase transformation occurring in a hydrothermal environment is of concern. In the present study, phase transformation and microstructure of tetragonal zirconia polycrystal partially stabilized with yttrium oxide (Y-TZP) and alumina-toughened zirconia (ATZ) sintered at different temperatures were estimated. On grazing angle X-ray diffraction analysis, ATZ showed less phase transformation to the monoclinic phase during hydrothermal treatment and this transformation appeared to occur within a few micrometers below the surface. At a higher sintering temperature the monoclinic phase content of ATZ was found to be lesser than that of Y-TZP, indicating that the alumina in ATZ was effective in suppressing hydrothermal degradation. Examination by transmission electron microscopy and studying of electron backscatter diffraction patterns indicated that grain growth in ATZ was slightly suppressed compared with that in Y-TZP at higher sintering temperatures. The present study demonstrated the effect of adding alumina to zirconia for suppressing hydrothermal degradation and studied the effect of this addition on grain growth in zirconia.