Investigation of a new implant surface modification using phosphorylated pullulan.

Various implant surface treatment methods have been developed to achieve good osseointegration in implant treatment. However, some cases remain impossible to treat with implants because osseointegration is not obtained after implantation, and the implants fail. Thus, this study focused on phosphorylated pullulan because of its adhesiveness to titanium (Ti) and bone, high biocompatibility, and early replacement with bone. In this study, the response of bone-related cells to phosphorylated pullulan was evaluated to develop a new surface treatment method. Saos-2 (human osteosarcoma-derived osteoblast-like cells), MC3T3-E1 (mouse calvaria-derived osteoblast-like cells), and RAW264.7 (mouse macrophage-like cells) were used. In evaluating cellular responses, phosphorylated pullulan was added to the culture medium, and cell proliferation and calcification induction tests were performed. The proliferation and calcification of cells on the surface of Ti disks coated with phosphorylated pullulan were also evaluated. In addition, bone morphogenetic protein-2 (BMP-2), an osteogenic factor, was used to evaluate the role of phosphorylated pullulan as a drug carrier in inducing calcification on Ti disks. Phosphorylated pullulan tended to promote the proliferation of osteoblast-like cells and the formation of calcification on Ti disks coated with phosphorylated pullulan. Ti disks coated with phosphorylated pullulan loaded with BMP-2 enhanced calcification. Phosphorylated pullulan inhibited osteoclast-like cell formation. These results are due to the properties of phosphorylated pullulan, such as adhesiveness to titanium and drug-loading function. Therefore, phosphorylated pullulan effectively promotes bone regeneration when coated on titanium implants and is useful for developing a new surface treatment method.

Epithelial-mesenchymal transition in oral cancer cells induced by prolonged and persistent Fusobacterium nucleatum stimulation.

OBJECTIVES: Several studies have reported the effects of Fusobacterium nucleatum stimulation on oral cancer cells. However, given that these studies typically span a stimulation period of three days to eight days, the in vitro studies conducted to date may not fully mimic the oral cancer environment, which involves constant exposure to oral commensal bacteria. This study aimed to elucidate the effects of prolonged and persistent Fusobacterium nucleatum infection on oral cancer cells. METHODS: Human tongue squamous cell carcinoma (SCC) cells were continuously stimulated with Fusobacterium nucleatum for two or four weeks, then experimentally evaluated. RESULTS: Prolonged, persistent Fusobacterium nucleatum stimulation increased the cells' proliferative, invasive, and migratory capacities, decreased their expression of epithelial markers, and increased their expression of mesenchymal markers progressively with time. The cells also adopted a spindle-shaped morphology and cell-to-cell contact dependence was progressively lost, suggesting time-dependent occurrence of epithelial-mesenchymal transition. Furthermore, mRNA levels of CD44, a cancer stem cell marker, were time-dependently upregulated. When SCC cells were stimulated with Fusobacterium nucleatum for four weeks in the presence of dexamethasone, Fusobacterium nucleatum induced epithelial-mesenchymal transition was inhibited. CONCLUSIONS: Epithelial-mesenchymal transition in human tongue SCC cells was time-dependently induced by prolonged, persistent Fusobacterium nucleatum stimulation and inhibited by dexamethasone. Routine decontamination of the oral cavity may be crucial for controlling tumor invasion and metastasis.

In vivo transmission electron microscopy of the alternating structure of the protrusions between adjacent macrophage-like cells on micropatterns.

OBJECTIVES: We investigated the behavior of macrophages in the defined microtopography of materials. METHODS: Patterned cyclo-olefin polymer films were implanted into the femurs of seven-week-old rats. After 1 and 4 weeks, the rats were fixed with glutaraldehyde and OsO4, and their bones were observed with transmission electron microscopy (TEM). RESULTS: TEM and segmentation revealed an alternating structure in which multiple protrusions of adjacent macrophage-like cells overlapped. They were approximately 2 µm long and almost uniform in width, and were induced by the limited topography. CONCLUSION: New structures appeared between the macrophage-like cells as a result of microtopography.

Can a low dosage of recombinant human bone morphogenetic protein-2 loaded on collagen sponge induce ectopic bone?

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is one of the growth factors that may induce the formation of new bone. The aim was to determine the efficacy of low doses of rhBMP-2 for bone regeneration using a collagen sponge as a carrier. Three doses of rhBMP-2 (1.167, 0.117, and 0.039 mg/mL) were combined with an absorbable collagen sponge (ACS) as a delivery vehicle. The rhBMP-2/ACS implants were placed in the subcutaneous tissues of rat backs. X-ray microcomputed tomography (micro-CT) and histological analysis were used to evaluate bone formation. The samples treated with 1.167 mg/mL of rhBMP-2 showed greater bone formation than the samples treated with 0.117 mg/mL of rhBMP-2 four weeks after surgery. However, there was no evidence of bone formation in the samples that were treated with 0.039 mg/mL of rhBMP-2. It was found that rhBMP-2 was osteogenic even at one-tenth of its manufacturer's recommended concentration (1.167 mg/mL), indicating its potential for clinical use at lower concentrations.

Periodontal tissue regeneration by recombinant human collagen peptide granules applied with ß-tricalcium phosphate fine particles.

OBJECTIVES: Recombinant human collagen peptide (RCP) is a recombinantly created xeno-free biomaterial enriched in arginine-glycine-aspartic acid sequences with good processability whose use for regenerative medicine applications is under investigation. The biocompatibility and osteogenic ability of RCP granules combined with ß-tricalcium phosphate (TCP) submicron particles (ß-TCP/RCP) were recently demonstrated. In the present study, ß-TCP/RCP was implanted into experimental periodontal tissue defects created in beagles to investigate its regenerative effects. METHODS: An RCP solution was lyophilized, granulated, and thermally cross-linked into particles approximately 1 mm in diameter. ß-TCP dispersion (1 wt%; 500 µL) was added to 100 mg of RCP granules to form ß-TCP/RCP. A three-walled intrabony defect (5 mm × 3 mm × 4 mm) was created on the mesial side of the mandibular first molar and filled with ß-TCP/RCP. RESULTS: A micro-computed tomography image analysis performed at 8 weeks postoperative showed a significantly greater amount of new bone after ß-TCP/RCP grafting (2.2-fold, P < 0.05) than after no grafting. Histological findings showed that the transplanted ß-TCP/RCP induced active bone-like tissue formation including tartaric acid-resistant acid phosphatase- and OCN-positive cells as well as bioabsorbability. Ankylosis did not occur, and periostin-positive periodontal ligament-like tissue formation was observed. Histological measurements performed at 8 weeks postoperative revealed that ß-TCP/RCP implantation formed 1.7-fold more bone-like tissue and 2.1-fold more periodontal ligament-like tissue than the control condition and significantly suppressed gingival recession and epithelial downgrowth (P < 0.05). CONCLUSIONS: ß-TCP/RCP implantation promoted bone-like and periodontal ligament-like tissue formation, suggesting its efficacy as a periodontal tissue regenerative material.

Osteoclast formation from mouse bone marrow cells on micro/nano-scale patterned surfaces.

OBJECTIVES: Osteoclasts can sense the surface topography of materials. However, it is difficult to identify the structural factors that affect osteoclast formation and its function. Furthermore, we hypothesized that the type of osteoclast precursor cells also affects osteoclastogenesis in the materials. In this study, we investigated the effects of defined micro/nanoscale patterns on osteoclastogenesis from bone marrow cells (BMCs). METHODS: Various cyclo-olefin polymer (COP) patterns were prepared using nanoimprinting. The effects of shape, size, and height of the patterns, and the wettability of the patterned surfaces on osteoclastogenesis from BMCs were evaluated in vitro. RESULTS: Osteoclast formation was promoted on pillars (diameter, 1 µm or 500 nm; height, 500 nm). Notably, osteoclastogenesis from BMCs was better promoted on hydrophobic pillars than on hydrophilic pillars. In contrast, decreased osteoclast formation was observed on the nanopillars (diameter, 100 nm; height, 200 nm). CONCLUSIONS: We demonstrated the promotion of osteoclast formation from BMCs on hydrophobic pillars with diameters of 1 µm and 500 nm. Some cellular behaviors in the patterns were dependent on the type of osteoclast precursor cells. The designed patterns are useful for designing the surface of dental implants or bone replacement materials with a controllable balance between osteoblast and osteoclast activities.

Bone forming ability of recombinant human collagen peptide granules applied with ß-tricalcium phosphate fine particles.

Recombinant human collagen peptide, developed based on human collagen type I, contains an arginyl-glycyl-aspartic acid (RGD)-rich motif to enhance cell behavior and is anticipated as a xeno-free polymer material for use in tissue engineering. We fabricated granules containing recombinant human collagen peptide (RCP) applied with beta-tricalcium phosphate fine particles (RCP/ß-TCP) as bone filling scaffold material and assessed the bone forming ability of RCP/ß-TCP. Recombinant peptide was thermal crosslinked and freeze-dried to prepare RCP. An aqueous dispersion of ß-TCP fine particles was added to RCP to obtain RCP/ß-TCP. Subsequently, RCP/ß-TCP were characterized using scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (EDX), and cell culture assessments. Furthermore, RCP/ß-TCP were implanted into rat cranial bone defects for radiographic and histological evaluations. In SEM and EDX analyses of RCP/ß-TCP, ß-TCP particles dose-dependently covered the surface of RCP. Cell culture tests showed that RCP/ß-TCP remarkably promoted proliferation and mRNA expression of various genes, such as integrin ß1 and osteogenic markers, of osteoblastic MC3T3-E1 cells. Histomorphometric assessment at 4 weeks showed that RCP/ß-TCP significantly promoted new skull bone formation compared to RCP (p < 0.05) and control (no application) (p < 0.01). Accordingly, these findings suggest RCP/ß-TCP possess bone forming capability and would be beneficial for bone tissue engineering therapy.

Proliferation of Saos-2 and Ca9-22 cells on grooved and pillared titanium surfaces.

BACKGROUND: Surface nanostructures in titanium (Ti) oral implants are critical for rapid osseointegration. OBJECTIVE: The purpose of this study was to evaluate the growth of osteoblast-like (Saos-2) and epithelial-like (Ca9-22) cells on nanopatterned Ti films. METHODS: Ti films with 500 nm grooves and pillars were fabricated by nanoimprinting, and seeded with Saos-2 and Ca9-22 cells. Cell viability and morphology were assessed by cell proliferation assay and scanning electron microscopy, respectively. RESULTS: As assessed after 1 hour, proliferation of Saos-2 cells was most robust on grooved films than on pillared and smooth films, in this order. These cells approximately doubled on grooved and pillared substrates in 24 hours and after 5 days, but not on smooth surfaces. In contrast, Ca9-22 cells favored smooth surfaces, followed by grooved and pillared films. Indeed, cells sparsely adhered to pillared films over 5 days of incubation (p < 0.05). CONCLUSIONS: The data show that Saos-2 and Ca9-22 cells respond differently to different nanostructures, and highlight the potential use of nanopatterns to promote bone regeneration or to prevent epithelial downgrowth at the implant-bone interface.

Antimicrobial photodynamic activity and cytocompatibility of Au25(Capt)18 clusters photoexcited by blue LED light irradiation.

Antimicrobial photodynamic therapy (aPDT) has beneficial effects in dental treatment. We applied captopril-protected gold (Au25(Capt)18) clusters as a novel photosensitizer for aPDT. Photoexcited Au clusters under light irradiation generated singlet oxygen (1O2). Accordingly, the antimicrobial and cytotoxic effects of Au25(Capt)18 clusters under dental blue light-emitting diode (LED) irradiation were evaluated. 1O2 generation of Au25(Capt)18 clusters under blue LED irradiation (420-460 nm) was detected by a methotrexate (MTX) probe. The antimicrobial effects of photoexcited Au clusters (0, 5, 50, and 500 µg/mL) on oral bacterial cells, such as Streptococcus mutans, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis, were assessed by morphological observations and bacterial growth experiments. Cytotoxicity testing of Au clusters and blue LED irradiation was then performed against NIH3T3 and MC3T3-E1 cells. In addition, the biological performance of Au clusters (500 µg/mL) was compared to an organic dye photosensitizer, methylene blue (MB; 10 and 100 µg/mL). We confirmed the 1O2 generation ability of Au25(Capt)18 clusters through the fluorescence spectra of oxidized MTX. Successful application of photoexcited Au clusters to aPDT was demonstrated by dose-dependent decreases in the turbidity of oral bacterial cells. Morphological observation revealed that application of Au clusters stimulated destruction of bacterial cell walls and inhibited biofilm formation. Aggregation of Au clusters around bacterial cells was fluorescently observed. However, photoexcited Au clusters did not negatively affect the adhesion, spreading, and proliferation of mammalian cells, particularly at lower doses. In addition, application of Au clusters demonstrated significantly better cytocompatibility compared to MB. We found that a combination of Au25(Capt)18 clusters and blue LED irradiation exhibited good antimicrobial effects through 1O2 generation and biosafe characteristics, which is desirable for aPDT in dentistry.

Estimation of Biocompatibility of Nano-Sized Ceramic Particles with Osteoblasts, Osteosarcomas and Hepatocytes by Static and Time-Lapse Observation.

We assessed the biocompatibility of nano-sized ceramic particles with several cells types. Though these particles have less than 100 nm in diameter, they act as submicron-sized particles in saline by aggregation that was estimated using laser diffraction particle size analysis (LDS). they act as submicro-sized particles in saline by aggregation based on laser diffraction particle size analysis (LDS). Several types of cells (osteoblasts, osteosarcoma and hepatocyte cells) were exposed to these particles and their cytocompatibility was estimated. Not only the cytotoxic assay but also their static and dynamic morphology under nanoparticles exposure were investigated. The intercellular uptake of particles was determined using a confocal fluorescence microscope. The particles used in this study did not inhibit cellular activity or growth even when their concentrations were high. Only copper oxide particles caused acute cytotoxicity depending on the particle size. The cytotoxicity assay, dynamic behavior of the nanoparticle-exposed cells and their examination under a confocal fluorescence microscope suggests that the irritative reaction was induced by contact between the cells and particles, whereas eluted copper ions are not dominant factor. These results indicate that nano-sized particles used in this study have excellent biocompatibility except copper oxide ones.

Comparative study of bioactivity of collagen scaffolds coated with graphene oxide and reduced graphene oxide.

BACKGROUND: Graphene oxide (GO) is a single layer carbon sheet with a thickness of less than 1 nm. GO has good dispersibility due to surface modifications with numerous functional groups. Reduced graphene oxide (RGO) is produced via the reduction of GO, and has lower dispersibility. We examined the bioactivity of GO and RGO films, and collagen scaffolds coated with GO and RGO. METHODS: GO and RGO films were fabricated on a culture dish. Some GO films were chemically reduced using either ascorbic acid or sodium hydrosulfite solution, resulting in preparation of RGO films. The biological properties of each film were evaluated by scanning electron microscopy (SEM), atomic force microscopy, calcium adsorption tests, and MC3T3-E1 cell seeding. Subsequently, GO- and RGO-coated collagen scaffolds were prepared and characterized by SEM and compression tests. Each scaffold was implanted into subcutaneous tissue on the backs of rats. Measurements of DNA content and cell ingrowth areas of implanted scaffolds were performed 10 days post-surgery. RESULTS: The results show that GO and RGO possess different biological properties. Calcium adsorption and alkaline phosphatase activity were strongly enhanced by RGO, suggesting that RGO is effective for osteogenic differentiation. SEM showed that RGO-modified collagen scaffolds have rough, irregular surfaces. The compressive strengths of GO- and RGO-coated scaffolds were approximately 1.7-fold and 2.7-fold greater, respectively, when compared with the non-coated scaffold. Tissue ingrowth rate was 39% in RGO-coated scaffolds, as compared to 20% in the GO-coated scaffold and 16% in the non-coated scaffold. CONCLUSION: In summary, these results suggest that GO and RGO coatings provide different biological properties to collagen scaffolds, and that RGO-coated scaffolds are more bioactive than GO-coated scaffolds.

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.

Toxicity evaluations of various carbon nanomaterials.

After the discovery of fullerene and carbon nanotubes, various carbon nanomaterials were discovered or synthesized. The carbon nanomaterials have remarkable properties, different from bulk materials with the same chemical composition, and are therefore useful for industrial applications. However, the toxicity of nanomaterials may also differ from that of the bulk materials; this difference poses a concern. The physical similarity of nanomaterials to asbestos has led to evaluations for toxicity by many researchers using various methods. In this review, we compile and compare the toxicity evaluations of each carbon nanomaterial.

3D collagen scaffolds coated with multiwalled carbon nanotubes: initial cell attachment to internal surface.

The cell adhesion in a multiwalled carbon nanotube-coated collagen sponge (MWCNT-coated sponge) was investigated. Immediately after seeding, the cells adhered to the inner surface of the MWCNT-coated sponge and a significantly larger number of cells were observed there than for a pure collagen sponge used as control. On the MWCNT-coated sponge, the cells appeared favorable adhesion and spread in the early stages in the center part of the sponge which cells rarely attached without MWCNT-coating. It was suggested that the physical structure of MWCNTs was effective for initial adhesion of cells from the result of serum-free culture. MWCNT-coating makes the material a suitable three-dimensional scaffold for cell culturing, as opposed to other scaffold systems where such an effect is not seen.

Material nanosizing effect on living organisms: non-specific, biointeractive, physical size effects.

Nanosizing effects of materials on biological organisms was investigated by biochemical cell functional tests, cell proliferation and animal implantation testing. The increase in specific surface area causes the enhancement of ionic dissolution and serious toxicity for soluble, stimulative materials. This effect originates solely from materials and enhances the same functions as those in a macroscopic size as a catalyst. There are other effects that become prominent, especially for non-soluble, biocompatible materials such as Ti. Particle size dependence showed the critical size for the transition of behaviour is at approximately 100 microm, 10 microm and 200 nm. This effect has its origin in the biological interaction process between both particles and cells/tissue. Expression of superoxide anions, cytokines tumour necrosis factor-alpha and interleukin-1 beta from neutrophils was increased with the decrease in particle size and especially pronounced below 10 microm, inducing phagocytosis to cells and inflammation of tissue, although inductively coupled plasma chemical analysis showed no dissolution from Ti particles. Below 200 nm, stimulus decreases, then particles invade into the internal body through the respiratory or digestive systems and diffuse inside the body. Although macroscopic hydroxyapatite, which exhibits excellent osteoconductivity, is not replaced with natural bone, nanoapatite composites induce both phagocytosis of composites by osteoclasts and new bone formation by osteoblasts when implanted in bone defects. The progress of this bioreaction results in the conversion of functions to bone substitution. Although macroscopic graphite is non-cell adhesive, carbon nanotubes (CNTs) are cell adhesive. The adsorption of proteins and nano-meshwork structure contribute to the excellent cell adhesion and growth on CNTs. Non-actuation of the immune system except for a few innate immunity processes gives the non-specific nature to the particle bioreaction and restricts reaction to the size-sensitive phagocytosis. Materials larger than cell size, approximately 10 microm, behave inertly, but those smaller become biointeractive and induce the intrinsic functions of living organisms. This bioreaction process causes the conversion of functions such as from biocompatibility to stimulus in Ti-abraded particles, from non-bone substitutional to bone substitutional in nanoapatite and from non-cell adhesive to cell adhesive CNTs. The insensitive nature permits nanoparticles that are less than 200 nm to slip through body defence systems and invade directly into the internal body.

In vitro evaluation of porous poly(L-lactic acid) scaffold reinforced by chitin fibers.

In this study, the previously reported porous three-dimensional poly(L-lactic acid) (PLLA) scaffolds reinforced by the chitin fibers (PLLA/CF) with and without the link were evaluated in vitro. Firstly, pH value of the phosphate buffered saline lixiviums of the PLLA/CF with different content of the chitin fibers was measured to get an appropriate content of the chitin fibers in the PLLA/CF. Then, the cell functions (attachment, proliferation, alkaline phosphatase per unit cell, total protein per unit cell, and osteonectin, osteopontin, and osteocalcin gene expression) of human osteoblast-like cells (SaOS2) cultured on the PLLA/CF with the link, PLLA/CF without the link and PLLA scaffold were compared. The results showed that the link treatment did not significantly influence the pH value of the lixiviums of the scaffolds, 30% volume content might be an appropriate content of the chitin fibers in PLLA/CF scaffold to keep the pH value of the lixiviums of the scaffolds between 7.0 and 7.2 during the lixiviation time of 16 weeks, the PLLA/CF scaffold was significantly better for the attachment, proliferation, differentiation, and mineralization of the osteoblast than PLLA, the link treatment did not significantly influence these cells activities, which further suggested that PLLA/CF with the link treatment might be an appropriate scaffold for tissue engineering.

Carbon nanotubes as scaffolds for cell culture and effect on cellular functions.

To investigate the dependence of biocompatibility of carbon materials on crystal structure with the aim of developing biomedical applications, single-(SW) and multi-walled (MW) carbon nanotubes (CNTs) were employed as scaffolds for cell culture and compared with graphite (GP). SaOS2 cells were used to investigate the properties and response of osteoblast-like cells. Polycarbonate membranes (PC) coated with CNTs by vacuum filtration formed a meshwork nanostructure. Cells grown on CNTs greatly extended in all directions. In terms of cell proliferation, alkaline phosphatase (ALP).activity, and protein adsorption on the substrates, CNTs showed better results than PC and GP. SW showed the best cell proliferation and total ALP. These favorable results might be attributed to the structure of CNTs and the affinity of CNTs toward proteins, thereby suggesting that CNTs could be potential scaffold materials for cell culture.

X-ray absorption fine structure (XAFS) analysis of titanium-implanted soft tissue.

Tissues contacting Ti dental implants were subjected to X-ray absorption fine structure (XAFS) analysis to examine the chemical state of Ti transferred from the placed implant into the surrounding tissue. Nine tissues that contacted pure Ti cover screws for several months were excised in a second surgery whereby healing abutments were set. Six tissues that surrounded implants retrieved due to their failure were also excised. Ti distributions in the excised specimens were confirmed by X-ray scanning analytical microscopy (XSAM), and the specimens were subjected to fluorescence XAFS analysis to determine the chemical states of the low concentrations of Ti in the tissues surrounding Ti dental implants. Ti mostly existed in the metallic state and was considered to be debris derived from the abrasion of implant pieces during implant surgery. Oxidized forms of Ti, such as anatase and rutile, were also detected in a few specimens-and existed in either a pure state or mixed state with metallic Ti. It was concluded that the existence of Ti in the tissue did not cause implant failure. Moreover, the usefulness of XAFS for analysis of the chemical states of rarely contained elements in biological tissue was demonstrated.

Human neutrophils reaction to the biodegraded nano-hydroxyapatite/collagen and nano-hydroxyapatite/collagen/poly(L-lactic acid) composites.

The impact of biodegraded nano-hydroxyapatite/collagen (nHAC) composite and nano-hydroxyapatite/collagen/poly(L-lactic acid) (nHAC/PLA) scaffold composite on neutrophils reaction was evaluated in vitro. Neutrophils were separated from human peripheral blood of healthy subjects. The nHAC and nHAC/PLA materials were immersed in the D-Hanks' Balanced Salt Solution (D-HBSS) for 1 day, 7 days and 2, 4, 8 weeks (37 degrees C) as testing solution, which mixed with the neutrophils for 1 h. Both of the nHAC and nHAC/PLA materials were shown the same cell survival rate as blank control, but the lactate dehydrogenase (LDH) and tumor necrosis factor alpha (TNF-alpha) released from the neutrophils were increased significantly after the 2 weeks in nHAC sample. The possible reason relied on the high concentration of calcium due to the quick biodegradation of the nHAC material. Before 2 weeks, the LDH value of nHAC/PLA is higher than that of nHAC sample that corresponded to the initial PLA degradation in vitro. This study provided the biocompatibility test of neutrophils other than common methods, such as osteoblastic cells for biomimetic materials. Moreover, it demonstrated the calcium concentration stimulating effect for cytokine release from neutrophils.

Strict preparation and evaluation of water-soluble hat-stacked carbon nanofibers for biomedical application and their high biocompatibility: influence of nanofiber-surface functional groups on cytotoxicity.

Water-soluble H-CNFs modified with a carboxyl group possessed the ability to induce TNF-alpha, whereas CHAPS-treated H-CNFs possessed significantly greater activity and were also found to activate NF-kappaB reporter activity, to a significantly greater level than H-CNFs; furthermore the functional group modified or coated on the surface of H-CNFs was a significant cytotoxic factor that affected cell activation.