A vitronectin-derived peptide prevents and restores alveolar bone loss by modulating bone re-modelling and expression of RANKL and IL-17A.

AIM: This study investigated whether a vitronectin-derived peptide (VnP-16) prevents and/or reverses alveolar bone resorption induced by ligature-induced periodontitis in rodents and identified the underlying mechanism. MATERIALS AND METHODS: We evaluated the effects of VnP-16 on osteogenic differentiation in human periodontal ligament cells (hPDLCs), lipopolysaccharide-induced inflammatory responses in gingival fibroblasts, and immune response in T lymphocytes. Ligature-induced periodontitis was induced by ligating the bilateral mandibular first molars for 14 days in rats and for 7 days in mice (n = 10/group). VnP-16 (100 µg/10 µl) was applied topically into the gingival sulcus of rats via intra-sulcular injection, whereas the peptide (50 µg/5 µl) was administered directly into the gingiva of mice via intra-gingival injection. To evaluate the preventive and therapeutic effects of VnP-16, micro-computed tomography analysis and histological staining were then performed. RESULTS: VnP-16 promoted osteogenic differentiation of periodontal ligament cells and inhibited the production of lipopolysaccharide-induced inflammatory mediators in gingival fibroblasts. Concomitantly, VnP-16 modulated the host immune response by reducing the number of receptor activator of NF-κB ligand (RANKL)-expressing lipopolysaccharide-stimulated CD4+ and CD8+ T cells, and by suppressing RANKL and interleukin (IL)-17A production. Furthermore, local administration of VnP-16 in rats and mice significantly prevented and reversed alveolar bone loss induced by ligature-induced periodontitis. VnP-16 enhanced osteoblastogenesis and simultaneously inhibited osteoclastogenesis and suppressed RANKL and IL-17A expression in vivo. CONCLUSIONS: Our findings suggest that VnP-16 acts as a potent therapeutic agent for preventing and treating periodontitis by regulating bone re-modelling and immune and inflammatory responses.

Toll-like receptor 9 mediates oral bacteria-induced IL-8 expression in gingival epithelial cells.

Previously, we reported that various oral bacteria regulate interleukin (IL)-8 production differently in gingival epithelial cells. The aim of this study was to characterize the pattern recognition receptor(s) that mediate bacteria-induced IL-8 expression. Among ligands that mimic bacterial components, only a Toll-like receptor (TLR) 9 ligand enhanced IL-8 expression as determined by ELISA. Both normal and immortalized human gingival epithelial (HOK-16B) cells expressed TLR9 intracellularly and showed enhanced IL-8 expression in response to CpG-oligonucleotide. The ability of eight strains of four oral bacterial species to induce IL-8 expression in HOK-16B cells, and their invasion capacity were examined in the absence or presence of 2% human serum. The ability of purified bacterial DNA (bDNA) to induce IL-8 was also examined. Six out of eight strains increased IL-8 production in the absence of serum. Usage of an endosomal acidification blocker or a TLR9 antagonist inhibited the IL-8 induction by two potent strains. In the presence of serum, many strains lost the ability to induce IL-8 and presented substantially reduced invasion capacity. The IL-8-inducing ability of bacteria in the absence or presence of serum showed a strong positive correlation with their invasion index. The IL-8-inducing ability of bacteria in the absence of human serum was also correlated with the immunostimulatory activity of its bDNA. The observed immunostimulatory activity of the bDNA could not be linked to its CpG motif content. In conclusion, oral bacteria induce IL-8 in gingival epithelial cells through TLR9 and the IL-8-inducing ability depends on the invasive capacity and immunostimulating DNA.

Treponema denticola suppresses expression of human {beta}-defensin-3 in gingival epithelial cells through inhibition of the toll-like receptor 2 axis.

We reported previously that Treponema denticola, one of the periodontal pathogens, suppresses the expression of human beta-defensins (HBDs) in human gingival epithelial cells. To identify the mechanisms involved in this suppression, immortalized and normal human gingival epithelial cells were infected with live or heat-killed T. denticola for 24 h, and then the expression of HBDs was examined by real-time RT-PCR. Live T. denticola suppressed the expression of HBD-3 substantially and also suppressed the expression of HBD-1 and HBD-2. However, heat-killed bacteria did not produce a suppressive effect but instead slightly upregulated the levels of HBD-2 and HBD-3. In contrast to live T. denticola, which reduced the activation of mitogen-activated protein kinase (MAPK) and NF-kappaB within an hour of infection, heat-killed bacteria did not show any inhibitory effect on the MAPK and NF-kappaB signaling pathways. Knockdown of Toll-like receptor 2 (TLR2) via RNA interference abolished the suppressive effect of T. denticola on the expression of HBD-3. Heat-killed T. denticola but not live bacteria could activate TLR2 in CHO/CD14/TLR2 reporter cells, suggesting that T. denticola contains a heat-labile inhibitor(s) of TLR2 in addition to ligands recognized by TLR2. Indeed, live T. denticola was able to inhibit TLR2 activation by Pam(3)CSK. In conclusion, T. denticola suppressed the expression of HBD-3 by inhibiting the TLR2 axis in gingival epithelial cells. These results may provide new insight into the pathogenesis of periodontitis caused by T. denticola.

A laminin-211-derived bioactive peptide promotes the osseointegration of a sandblasted, large-grit, acid-etched titanium implant.

Early implant loading is very important for reducing the duration of missing teeth in human patients. The laminin-derived peptide, DLTIDDSYWYRI motif (Ln2-P3), accelerates bone healing. Therefore, to investigate the hypothesis that Ln2-P3 increases the bone response to sandblasted, large-grit, acid-etched (SLA) titanium implants, the effect of the Ln2-P3 peptide on the osseointegration of SLA titanium implants was evaluated in vitro and in vivo. Human osteoblast-like cells were cultured on untreated, scrambled peptide (SP)-treated, and Ln2-P3-treated SLA titanium discs, and the cellular responses of these cells were evaluated. The Ln2-P3 treatment augmented osteoblast attachment and spreading, alkaline phosphatase activity, and the expression of osteogenic marker genes. Furthermore, the untreated and Ln2-P3-treated SLA titanium implants were inserted into the tibiae of rabbits for 9 and 11 days. Compared with the untreated implants, the Ln2-P3-treated implants showed a significantly higher bone-to-implant contact ratio at Day 9 after implantation and an increased bone area. The Ln2-P3 treatment of the SLA titanium implant surface augmented osteoblastic activity and accelerated peri-implant bone formation at the bone-implant interface. Overall, these results indicated that compared with the SLA titanium surface alone, the Ln2-P3 peptide-treated SLA titanium surface enhances initial osseointegration, thereby facilitating earlier implant loading.

Toll-like receptor 2 and NALP2 mediate induction of human beta-defensins by fusobacterium nucleatum in gingival epithelial cells.

We previously reported that infection by Fusobacterium nucleatum strongly induced the expression of both human beta-defensin 2 (HBD-2) and HBD-3 by gingival epithelial cells. The aim of this study was to characterize the pattern recognition receptors (PRRs) and regulatory mechanisms involved in the induction of HBD-2 and -3 expression by F. nucleatum in gingival epithelial cells. Immortalized human gingival epithelial HOK-16B cells were infected with live or heat-killed F. nucleatum, and the expression of HBDs and interleukin-8 (IL-8) was examined by real-time reverse transcription-PCR and enzyme-linked immunosorbent assay, respectively. Live, but not heat-killed, F. nucleatum invaded HOK-16B cells, as seen by confocal microscopy and flow cytometry. Live F. nucleatum induced both HBD-2 and -3 efficiently, whereas heat-killed bacteria induced only HBD-3 at a reduced level. Knockdown of NACHT-LRR- and pyrin domain-containing protein 2 (NALP2), the most abundant intracellular PRR in HOK-16B cells, by RNA interference (RNAi) significantly reduced the induction of HBD-3 but not HBD-2 and IL-8. In addition, knockdown of Toll-like receptor 2 (TLR2) by RNAi reduced the upregulation of HBD-2 and -3 but not IL-8. Heat-killed F. nucleatum was hindered in its ability to activate TLR2 and JNK signaling pathways. Theses data show that TLR2 and NALP2 mediate the induction of HBDs by F. nucleatum in gingival epithelial cells, but thresholds for the two HBD genes are different.

A Vitronectin-Derived Bioactive Peptide Improves Bone Healing Capacity of SLA Titanium Surfaces.

In this study, we evaluated early bone responses to a vitronectin-derived, minimal core bioactive peptide, RVYFFKGKQYWE motif (VnP-16), both in vitro and in vivo, when the peptide was treated on sandblasted, large-grit, acid-etched (SLA) titanium surfaces. Four surface types of titanium discs and of titanium screw-shaped implants were prepared: control, SLA, scrambled peptide-treated, and VnP-16-treated surfaces. Cellular responses, such as attachment, spreading, migration, and viability of human osteoblast-like HOS and MG63 cells were evaluated in vitro on the titanium discs. Using the rabbit tibia model with the split plot design, the implants were inserted into the tibiae of four New Zealand white rabbits. After two weeks of implant insertion, the rabbits were sacrificed, the undecalcified specimens were prepared for light microscopy, and the histomorphometric data were measured. Analysis of variance tests were used for the quantitative evaluations in this study. VnP-16 was non-cytotoxic and promoted attachment and spreading of the human osteoblast-like cells. The VnP-16-treated SLA implants showed no antigenic activities at the interfaces between the bones and the implants and indicated excellent bone-to-implant contact ratios, the means of which were significantly higher than those in the SP-treated implants. VnP-16 reinforces the osteogenic potential of the SLA titanium dental implant.

A laminin-derived functional peptide, PPFEGCIWN, promotes bone formation on sandblasted, large-grit, acid-etched titanium implant surfaces.

PURPOSE: This study aimed to investigate the in vitro and in vivo bone-forming potential of a sandblasted, large-grit, acid-etched (SLA) titanium (Ti) surface treated with a laminin-derived functional peptide, PPFEGCIWN (DN3). MATERIALS AND METHODS: Human osteoblast-like MG63 cells were cultured with SLA Ti discs untreated or treated with DN3 or a control scrambled peptide (SP). Cell adhesion, spreading, and viability on the discs were tested. Alkaline phosphatase gene expression and enzyme activity were also evaluated. Four DN3-coated SLA Ti implants and four untreated implants were placed into the tibiae of two rabbits (two implants/tibia). Ten days later, the bone-implant interfaces were subjected to histomorphometry to measure the bone response. The surface properties of the discs and implants were determined using scanning electron, widefield confocal, and confocal laser microscopy and x-ray photoelectron spectroscopy. RESULTS: The peptide-treated and untreated discs and implants were similar in terms of physical surface properties, but the peptide-treated surfaces had significantly higher nitrogen levels (P < .05). The DN3 peptide promoted cell adhesion, spreading, and alkaline phosphatase expression and enzyme activity (P < .05). Histomorphometry of the harvested implants showed rapid bone formation and affinity of the motif. CONCLUSION: This study suggests that treatment with the cell adhesion peptide DN3 promotes bone healing at the SLA Ti surface.

Osteogenic potential of embryonic stem cells in tooth sockets.

Embryonic stem cells (ESCs) are established from blastocysts and give rise to various types of cells and tissues. In the present study, we assessed the osteogenic potential of ESCs using in vitro culture conditions and in vivo differentiation in tooth sockets. An ESC-derived embryoid body (EB) was formed and subsequently induced to an osteogenic lineage. The differentiated EB cells exhibited increased expression of various osteogenic markers as determined by real-time PCR analysis. Likewise, the differentiated EB-derived cells had enhanced alkaline phosphatase activity and calcium accumulation, as determined by cytochemical methods. For in vivo transplantation, mixtures of ESCs and hydroxyapatite/ tricalcium phosphate particles or EBs alone were transplanted into female rat tooth sockets. After 12 weeks, we observed formation of osteogenic structure in the tooth sockets without evidence of teratomas. These data suggest that pluripotent ESCs can serve as an alternative source for the reconstruction of craniofacial structures, as well as for further applications.

Effect of chitin/silk fibroin nanofibrous bicomponent structures on interaction with human epidermal keratinocytes.

To fabricate a biomimetic nanostructured bicomponent scaffolds, two types of chitin/silk fibroin (SF) nanofibrous scaffolds (blend scaffolds and hybrid scaffolds) were prepared by electrospinning or simultaneous electrospinning of chitin/SF solutions. The chitin/SF bicomponent scaffolds were after-treated with water vapor, and their nanofibrous structures were almost maintained. From the cytocompatibility and cell behavior on the chitin/SF blend or hybrid nanofibrous scaffolds, the hybrid matrix with 25% chitin and 75% SF as well as the chitin/SF blend nanofibers could be a potential candidate for tissue engineering scaffolds.

A vitronectin-derived peptide reverses ovariectomy-induced bone loss via regulation of osteoblast and osteoclast differentiation.

Osteoporosis affects millions of people worldwide by promoting bone resorption and impairing bone formation. Bisphosphonates, commonly used agents to treat osteoporosis, cannot reverse the substantial bone loss that has already occurred by the time of diagnosis. Moreover, their undesirable side-effects, including osteonecrosis of the jaw, have been reported. Here, we demonstrated that a new bioactive core vitronectin-derived peptide (VnP-16) promoted bone formation by accelerating osteoblast differentiation and activity through direct interaction with ß1 integrin followed by FAK activation. Concomitantly, VnP-16 inhibited bone resorption by restraining JNK-c-Fos-NFATc1-induced osteoclast differentiation and αvß3 integrin-c-Src-PYK2-mediated resorptive function. Moreover, VnP-16 decreased the bone resorbing activity of pre-existing mature osteoclasts without changing their survival rate. Furthermore, VnP-16 had a strong anabolic effect on bone regeneration by stimulating osteoblast differentiation and increasing osteoblast number, and significantly alleviated proinflammatory cytokine-induced bone resorption by restraining osteoclast differentiation and function in murine models. Moreover, VnP-16 could reverse ovariectomy-induced bone loss by both inhibiting bone resorption and promoting bone formation. Given its dual role in promoting bone formation and inhibiting bone resorption, our results suggest that VnP-16 could be an attractive therapeutic agent for treating osteoporosis.

Biocompatibility of nanocomposites used for artificial conjunctiva: in vivo experiments.

PURPOSE: To evaluate the biocompatibility of nanocomposites used for artificial conjunctiva. METHODS: Fifty New Zealand white rabbits were used for the experiments. Nanocomposites of poly -caprolactone (PCL) and of PCL coated with polyvinyl alcohol (PCL+PVA), polyvinyl pyrrolidone (PCL+PVP), or chitosan (PCL+C), and amniotic membrane (AM) as a control, were cut into small disks with a diameter of 3.5 mm. The disks were inserted underneath the conjunctiva to measure their inflammation-inducing properties. To investigate epithelial adhesion and goblet cell differentiation, the disks were transplanted after round conjunctival excision. Cultivated conjunctival epithelial cells on nanocomposite were then transplanted onto the abdomen of Balb/c athymic mice. The number of inflammatory cells and the density of goblet cells were measured using hematoxylin and eosin, periodic-acid-Schiff, and immunohistochemistry after 2 weeks. RESULTS: The number of inflammatory cells found inside of the inserts was as follows: 21 +/- 4.9 for controls, 21 +/- 15.1 for PCL, 49.6 +/- 26.0 for PCL+PVP, 40.2 +/- 17.1 for PCL+C, and 13.8 +/- 3.9 for PCL+PVA. In PCL+PVA, the accumulation of inflammatory cells was significantly lower than in the controls (p < 0.01, Mann-Whitney U). The reepithelialization of conjunctival cells was accomplished in more than 75% of all disks except for the PCL+C. In addition, we found the differentiation of goblet cells in the following order from greatest to least: amniotic membrane, PCL, and PCL+PVP. CONCLUSIONS: Nanocomposites of PCL were biocompatible in rabbit conjunctiva, suggesting that PCL may be considered as a candidate for use in the development of artificial conjunctiva.

Electrospinning of collagen nanofibers: effects on the behavior of normal human keratinocytes and early-stage wound healing.

Electrospinning of type I collagen in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to fabricate a biomimetic nanofibrous extracellular matrix for tissue engineering was investigated. The average diameter of collagen nanofibers electrospun from 8% collagen solution in HFIP was 460 nm (range of 100-1200 nm). The as-spun collagen nanofibrous matrix was chemically cross-linked by glutaraldehyde vapor with a saturated aqueous solution and then treated with aqueous 0.1m glycine to block unreacted aldehyde groups. With vapor phase cross-linking for 12h, porosity of the collagen matrix decreased from 89% to 71%. The collagen nanofibrous matrix showed good tensile strength, even in aqueous solution. Effects on cytocompatibility, cell behavior, cell and collagen nanofiber interactions, and open wound healing in rats were examined. Relatively low cell adhesion was observed on uncoated collagen nanofibers, whereas collagen nanofibrous matrices treated with type I collagen or laminin were functionally active in responses in normal human keratinocytes. Collagen nanofibrous matrices were very effective as wound-healing accelerators in early-stage wound healing. Our results indicate that cross-linked collagen nanofibers coated with ECM proteins, particularly type I collagen, may be a good candidate for biomedical applications, such as wound dressing and scaffolds for tissue engineering.

First isolation of Streptococcus downei from human dental plaques.

In this study, we isolated four bacterial strains grown on mitis-salivarius sucrose bacitracin agar. The strains had similar biochemical characteristics to biotypes I or II of mutans streptococci. The four isolates were identified as Streptococcus downei by 16S rDNA and dextranase gene (dex) sequencing as well as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) targeting dex. To our knowledge, this is the first report of the isolation and identification of S. downei from dental plaque in humans. The results suggest that S. downei can inhabit the human oral cavity.

Effects of adhesion molecules on the behavior of osteoblast-like cells and normal human fibroblasts on different titanium surfaces.

This study examined the influences of titanium (Ti) discs with similar surface roughnesses (R(a) values), but with different topographies and chemical compositions, on the adhesion, spreading, and the alkaline phosphatase (ALP) activity of osteoblast-like cells and normal human fibroblasts. The presence of adhesion molecules on the Ti surfaces and their effects on cell activity were also investigated. Two types of Ti discs were prepared. One kind was a mechanically polished Ti disc, and the other type was a disc obtained by the heating of hydroxyapatite (HA) dip-coated Ti. Scanning electron microscopy, optical interferometry, and scanning Auger electron spectroscopy were used to examine the surface morphology, roughness, and chemical composition, respectively, of the superficial Ti layer. The two types of Ti discs had different topographies and chemical compositions, but had similar R(a) values. The cells on both surface types had similar behaviors and ALP activities. A biological evaluation of the surface-modified Ti discs showed that the type I collagen coating was functionally active in terms of cell spreading in both types of Ti discs. In the mechanically polished Ti discs, fibronectin was functionally active in the normal human fibroblasts, but not in the osteoblast-like cells. Cell adhesion was slightly better on the heat-treated HA dip-coated Ti discs, but not on the mechanically polished Ti discs. Type I collagen and fibronectin mediated the adhesion and spreading of osteoblast-like cells through alpha2beta1 integrin and alpha5beta1 integrin, respectively. These results suggest that type I collagen might be a good candidate for the biochemical modification of Ti surfaces, particularly those surfaces obtained by heating of HA dip-coated Ti.

Identification of non-mutans streptococci organisms in dental plaques recovering on mitis-salivarius bacitracin agar medium.

The objective of this study was to both isolate and identify non-mutans streptococci organisms (non-MSO) from dental plaques recovered on mitis-salivarius sucrose bacitracin agar (MSB) plates. The dental plaque samples, which had been collected from 63 human subjects, were diluted and plated on MSB. The bacteria growing on the MSB plates were then identified with biochemical tests, as well as with 16S rDNA cloning and sequencing techniques. Our data indicated that bacteria from 30 subjects had been recovered on the MSB plates. Among the 21 typical colonies selected from the 30 subjects, 12 colonies, derived from 10 subjects, were identified as non-MSO. These 12 colonies were determined to be Streptococcus anginosus (8 colonies), S. sanguinis (1 colony), and Pantoea agglomerans (3 colonies). These results strongly suggest that a new selective medium will be required for the reliable isolation of mutans streptococci.

Characterization of novel cell lines established from three human oral squamous cell carcinomas.

Human oral squamous cell carcinoma cell lines (KOSCC-11, -25A, -25B, -25C, -25D, -25E, -33A, and -33B) were established by explantation culture from these oral squamous cell carcinomas. The histopathology of the primary tumors, in vitro growth characteristics, epithelial origin, in vitro anchorage-independency, in vivo tumorigenicity, the frequency of human papillomavirus (HPV) infections, and the status of proto-oncogenes, tumor suppressor genes, DNA mismatch repair genes, and microsatellite instability were investigated in the cell lines. KOSCC-11 is a well-differentiated oral squamous cell carcinoma (OSCC) derived from mandibular gingiva. KOSCC-25A, -25B, -25C, -25D, and -25E cell lines were derived from the same OSCC. KOSCC-33A and -33B were established from the same tumor that originated from the maxillary sinus. All tumor lines studied grew as monolayers and showed: i) epithelial origin by the presence of desmosome and keratin; ii) in vitro anchorage-independent growth ability; and iii) tumorigenic potential in nude mice. The cancer cell lines did not contain HPV DNA and did not express viral genes. Northern blot analysis revealed: i) overexpression of EGFR in four cell lines, ii) overexpression of c-H-ras in four cell lines, iii) overexpression of c-myc in three cell lines, iv) decreased expression of TGF-alpha in seven cell lines, and v) decreased expression of c-jun in five cancer cell lines compared with normal human oral keratinocytes. In all KOSCC cell lines and their corresponding tumor tissues, mutations were identified in highly-conserved functional regions of the p53 gene. The KOSCC-11 cell line contained a frameshift mutation and the other cell lines harbored an identical p53 mutation at codon 175 from CGC (Arg) to CTC (Leu). In five cell lines, a significant reduction of p21WAF1/Cip1 protein was evident. Cancer cell lines expressed higher level of Rb protein than normal human oral keratinocytes. DCC, a tumor suppressor gene, was not detected in KOSCC-25C. The KOSCC-33A cell line displayed microsatellite instability and showed a loss of hMSH2 expression. These well-characterized human OSCC cell lines should serve as useful tools for understanding the biological characteristics of oral cancer.

Electrospinning of silk fibroin nanofibers and its effect on the adhesion and spreading of normal human keratinocytes and fibroblasts in vitro.

An electrospinning method was used to fabricate silk fibroin (SF) nanofiber nonwovens for cell culture of normal human keratinocytes and fibroblasts. The electrospinning of regenerated SF was performed with formic acid as a spinning solvent. For insolubilization, as-spun SF nanofiber nonwovens were chemically treated with an aqueous methanol solution of 50%. Morphology and microstructure of as-spun and chemically treated SF nanofibers were investigated by scanning electron microscopy and mercury porosimetry. As-spun SF nanofibers exhibited a circular cross-section with a smooth surface. From the image analysis, they had an average diameter of 80 nm and their diameters ranged from 30 to 120 nm. During the chemical treatment for 60 min, porosity of nonwovens composed of SF nanofibers decreased from 76.1% up to 68.1%. To assay the cytocompatibility and cell behavior onto the electrospun SF nanofibers, cell attachment and spreading of normal human keratinocytes and fibroblasts seeded on the SF nanofibers and interaction between cells and SF nanofibers were studied. Cell morphology on SF nanofibers was examined by scanning electron microscopy. Our results indicate that the SF nanofibers may be a good candidate for the biomedical applications, such as wound dressing and scaffolds for tissue engineering.

Determination of specific interactions between glucose ligand carrying polymer and glucose transporter type-1 (GLUT-1) using different cell types.

In order to develop a biomimetic polymer for cell recognition, poly [3-O-(4'-vinylbenzyl)-D-glucose] (PVG) and different types of glucose transport (GLUT)-carrying cells, namely, HepG2 cells (GLUT-1), 3T3-L1 fibroblast cells (GLUT-1 and GLUT-4), and MIN6 cells (GLUT-2), were tested for specific interaction. To clarify the nature of interaction between PVG and the different cell types, rhodamine-B isothiocyanate (RITC)-labeled polymers were used to prove and visualize the interactions. In this study, we found that labeled polymer strongly binds to HepG2 cells. The fluorescence intensity of PVG with in the presence of HepG2 cells was found to be stronger than that of 3T3-L1 fibroblast cells (0.11 +/- 0.05) and MIN6 cells, which carry GLUT-2 (0.028 +/- 0.01); a confocal laser microscopic study confirmed this interaction. To confirm the specificity of the interaction mediated by GLUT, the cells were pretreated with phloretin, an inhibitor of GLUT-1, before adding RITC-labeled PVG polymer to the cell culture medium. This treatment suppressed the interaction of PVG with HepG2 cells and partially suppressed its interaction with 3T3-L1 fibroblast cells.

Titanium surface coating with a laminin-derived functional peptide promotes bone cell adhesion.

Laminin-derived peptide coatings can enhance epithelial cell adhesion to implants, and the positive effect of these peptides on bone cell adhesion has been anticipated. The purpose of this study was to evaluate the improvement in bone cell attachment to and activity on titanium (Ti) scaffolds coated with a laminin-derived functional peptide, Ln2-P3 (the DLTIDDSYWYRI motif). Four Ti disc surfaces were prepared, and a human osteosarcoma (HOS) cell attachment test was performed to select two candidate surfaces for peptide coating. These two candidates were then coated with Ln2-P3 peptide, a scrambled peptide, or left uncoated to measure cell attachment to each surface, following which one surface was chosen to assess alkaline phosphatase (ALP) activity and osteogenic marker gene expression with quantitative real-time PCR. On the commercially pure Ti surface, the Ln2-P3 coating significantly increased cellular ALP activity and the expression levels of ALP and bone sialoprotein mRNA as compared with the scrambled peptide-coated and uncoated surfaces. In conclusion, although further in vivo studies are needed, the findings of this in vitro study indicate that the Ln2-P3-coated implant surface promotes bone cell adhesion, which has clinical implications for reducing the overall treatment time of dental implant therapy.

The effect of the DLTIDDSYWYRI motif of the human laminin α2 chain on implant osseointegration.

Considerable effort has been directed towards replacing lost teeth using tissue-engineering methods such as titanium implants. A number of studies have tried to modify bioinert titanium surfaces by coating them with functionally bioactive molecules for faster and stronger osseointegration than pure titanium surfaces. Recently, peptides have been recognized as valuable scientific tools in the field of tissue-engineering. The DLTIDDSYWYRI motif of the human laminin-2 α2 chain has been previously reported to promote the attachment of various cell types; however, the in vivo effects of the DLTIDDSYWYRI motif on new bone formation have not yet been studied. To examine whether a laminin-2-derived peptide can promote osseointegration by accelerating new bone formation in vivo, we applied titanium implants coated with the DLTIDDSYWYRI motif in a rabbit tibia model. The application of the DLTIDDSYWYRI motif-treated implant to tibia wounds enhanced collagen deposition and alkaline phosphatase expression. It significantly promoted implant osseointegration compared with treatment with scrambled peptide-treated implants by increasing the bone-to-implant contact ratio and bone area. These findings support the hypothesis that the DLTIDDSYWYRI motif acts as an effective osseointegration accelerator by enhancing new bone formation.