Structural and Chemical Hierarchy in Hydroxyapatite Coatings.

Hydroxyapatite coatings need similarly shaped splats as building blocks and then a homogeneous microstructure to unravel the structural and chemical hierarchy for more refined improvements to implant surfaces. Coatings were thermally sprayed with differently sized powders (20-40, 40-63 and 63-80 µm) to produce flattened homogeneous splats. The surface was characterized for splat shape by profilometry and Atomic force microscopy (AFM), crystal size by AFM, crystal orientation by X-ray diffraction (XRD) and structural variations by XRD. Chemical composition was assessed by phase analysis, but variations in chemistry were detected by XRD and Raman spectroscopy. The resulting surface electrical potential was measured by Kelvin probe AFM. Five levels of structural hierarchy were suggested: the coating, the splat, oriented crystals, alternate layers of oxyapatite and hydroxyapatite (HAp) and the suggested anion orientation. Chemical hierarchy was present over a lower range of order for smaller splats. Coatings made from smaller splats exhibited a greater electrical potential, inferred to arise from oxyapatite, and supplemented by ordered OH- ions in a rehydroxylated surface layer. A model has been proposed to show the influence of structural hierarchy on the electrical surface potential. Structural hierarchy is proposed as a means to further refine the properties of implant surfaces.

Friction force microscopy of tribochemistry and interfacial ageing for the SiO x /Si/Au system.

Friction force microscopy was performed with oxidized or gold-coated silicon tips sliding on Au(111) or oxidized Si(100) surfaces in ultrahigh vacuum. We measured very low friction forces compared to adhesion forces and found a modulation of lateral forces reflecting the atomic structure of the surfaces. Holding the force-microscopy tip stationary for some time did not lead to an increase in static friction, i.e., no contact ageing was observed for these pairs of tip and surface. Passivating layers from tip or surface were removed in order to allow for contact ageing through the development of chemical bonds in the static contact. After removal of the passivating layers, tribochemical reactions resulted in strong friction forces and tip wear. Friction, wear, and the re-passivation by oxides are discussed based on results for the temporal development of friction forces, on images of the scanned area after friction force microscopy experiments, and on electron microscopy of the tips.

Suture materials affect peri-implant bone healing and implant osseointegration.

The aim of this study was to evaluate the effects of the remnants of two suture materials on osseointegration of titanium implants in a rabbit tibial model. Calibrated defects were prepared in the tibia of five Chinchilla rabbits. Filaments of nonresorbable (NR) nylon or resorbable (R) chitosan were placed at the bone to implant interface, whereas control sites had no suture material. After a healing period of 4 weeks, a pull-out test procedure was performed followed by enzymatic analyses of the wound fluid and relative quantification of mRNA levels for bone-related and cytokine markers from the peri-implant bone. A trend toward a reduced pull-out force was observed in the NR group (NR: 23.0 ± 12.8 N; R: 33.9 ± 11.3 N; control: 33.6 ± 24.0 N). Similarly, the bone resorption marker vacuolar type H+-ATPase was increased in the NR group compared with that in the control group (P = 0.041). The R group showed trends for lower alkaline phosphatase activity and osteocalcin expression and higher total protein content and RNA compared with the control group. In this submerged healing model, peri-implant bone healing was marginally affected by the two suture materials tested. However, there was a tendency toward better osseointegration and lower expression of bone resorption markers in the R group compared with the control group.

Proline-Rich Peptide Mimics Effects of Enamel Matrix Derivative on Rat Oral Mucosa Incisional Wound Healing.

BACKGROUND: Proline-rich peptides have been shown to promote periodontal regeneration. However, their effect on soft tissue wound healing has not yet been investigated. The aim of this study is to evaluate the effect of enamel matrix derivative (EMD), tyrosine-rich amelogenin peptide (TRAP), and a synthetic proline-rich peptide (P2) on acute wound healing after a full-thickness flap procedure in an incisional rat model. METHODS: This experimental study has a split-mouth, randomized, placebo-controlled design. Test and control wounds were created on the palatal mucosa of 54 Sprague-Dawley rats. Wounds were histologically processed, and reepithelialization, leukocyte infiltration, and angiogenesis were assessed at days 1, 3, and 7 post-surgery. RESULTS: EMD and P2 significantly promoted early wound closure at day 1 (P <0.001 and P = 0.004, respectively). EMD maintained a significant acceleration of reepithelialization at day 3 (P = 0.004). Wounds treated by EMD and P2 showed increased angiogenesis during the first 3 days of healing (P = 0.03 and 0.001, respectively). Leukocyte infiltration was decreased in EMD-treated wounds at day 1 (P = 0.03), and P2 and TRAP induced a similar effect at days 3 (P = 0.002 and P <0.0001, respectively) and 7 (P = 0.005 and P <0.001). CONCLUSION: EMD and P2 promoted reepithelialization and neovascularization in full-thickness surgical wounds on rat oral mucosa.

Flavonoid-modified surfaces: multifunctional bioactive biomaterials with osteopromotive, anti-inflammatory, and anti-fibrotic potential.

Flavonoids are small polyphenolic molecules of natural origin with antioxidant, anti-inflammatory, and antibacterial properties. Here, a bioactive surface based on the covalent immobilization of flavonoids taxifolin and quercitrin on titanium substrates is presented, using (3-aminopropyl)triethoxysilane (APTES) as coupling agent. FTIR and XPS measurements confirm the grafting of the flavonoids to the surfaces. Using 2-aminoethyl diphenylborinate (DPBA, a flavonoid-specific dye), the modified surfaces are imaged by fluorescence microscopy. The bioactivity of the flavonoid-modified surfaces is evaluated in vitro with human umbilical cord derived mesenchymal stem cells (hUC-MSCs) and human gingival fibroblasts (HGFs) and compared to that of simple flavonoid coatings prepared by drop casting. Flavonoid-modified surfaces show anti-inflammatory and anti-fibrotic potential on HGF. In addition, Ti surfaces covalently functionalized with flavonoids promote the differentiation of hUC-MSCs to osteoblasts--enhancing the expression of osteogenic markers, increasing alkaline phosphatase activity and calcium deposition; while drop-casted surfaces do not. These findings could have a high impact in the development of advanced implantable medical devices like bone implants. Given the broad range of bioactivities of flavonoid compounds, these surfaces are ready to be explored for other biomedical applications, e.g., as stent surface or tumor-targeted functionalized nanoparticles for cardiovascular or cancer therapies.

The skeletal phenotype of chondroadherin deficient mice.

Chondroadherin, a leucine rich repeat extracellular matrix protein with functions in cell to matrix interactions, binds cells via their α2ß1 integrin as well as via cell surface proteoglycans, providing for different sets of signals to the cell. Additionally, the protein acts as an anchor to the matrix by binding tightly to collagens type I and II as well as type VI. We generated mice with inactivated chondroadherin gene to provide integrated studies of the role of the protein. The null mice presented distinct phenotypes with affected cartilage as well as bone. At 3-6 weeks of age the epiphyseal growth plate was widened most pronounced in the proliferative zone. The proteome of the femoral head articular cartilage at 4 months of age showed some distinct differences, with increased deposition of cartilage intermediate layer protein 1 and fibronectin in the chondroadherin deficient mice, more pronounced in the female. Other proteins show decreased levels in the deficient mice, particularly pronounced for matrilin-1, thrombospondin-1 and notably the members of the α1-antitrypsin family of proteinase inhibitors as well as for a member of the bone morphogenetic protein growth factor family. Thus, cartilage homeostasis is distinctly altered. The bone phenotype was expressed in several ways. The number of bone sialoprotein mRNA expressing cells in the proximal tibial metaphysic was decreased and the osteoid surface was increased possibly indicating a change in mineral metabolism. Micro-CT revealed lower cortical thickness and increased structure model index, i.e. the amount of plates and rods composing the bone trabeculas. The structural changes were paralleled by loss of function, where the null mice showed lower femoral neck failure load and tibial strength during mechanical testing at 4 months of age. The skeletal phenotype points at a role for chondroadherin in both bone and cartilage homeostasis, however, without leading to altered longitudinal growth.

Effect of proline-rich synthetic peptide-coated titanium implants on bone healing in a rabbit model.

PURPOSE: Previous studies have demonstrated the capacity of a designed proline-rich synthetic peptide to stimulate osteoblast differentiation and biomineralization in vitro. Therefore, the aim of the present study was to evaluate the osseointegration capacity of titanium (Ti) implants coated with these peptides in a rabbit model. MATERIALS AND METHODS: Four calibrated defects were prepared in the tibiae of three New Zealand rabbits, and the defects were randomized into a test group (peptide-modified machined Ti implant) and a control group (unmodified machined Ti implant). The performance in vivo was investigated after 4 weeks of implantation by real-time reverse transcriptase polymerase chain reaction of bone and inflammatory markers, microcomputed tomographic analysis of mineralized bone, and histologic examination. RESULTS: The peptides adsorbed in agglomerates on Ti and underwent a change in secondary structure upon adsorption, which induced an increase in surface wettability. Gene expression markers indicated that peptide-coated Ti implants had significantly decreased mRNA levels of tartrate-resistant acid phosphatase. A trend toward increased osteocalcin in the peri-implant bone tissue was also seen. Bone morphometric and histologic parameters did not show significant differences, although the peptide group showed a higher percentage of new bone histologically. CONCLUSIONS: Proline-rich peptides have potential as a biocompatible coating for promoting osseointegration of Ti implants by reducing bone resorption.

Activation of dendritic cells by the novel Toll-like receptor 3 agonist RGC100.

Toll-like receptor (TLR) 3 agonists emerged as attractive candidates for vaccination strategies against tumors and pathogens. An important mechanism of action of such agonists is based on the activation of TLR3-expressing dendritic cells (DCs), which display a unique capacity to induce and stimulate T-cell responses. In this context, it has been demonstrated that targeting of TLR3 by double-stranded RNA such as poly(I:C) results in potent activation of DCs. Major disadvantages of poly(I:C) comprise its undefined chemical structure and very poor homogeneity, with subsequent unpredictable pharmacokinetics and high toxicity. In the present study, we evaluated the physicochemical properties and biological activity of the novel TLR3 agonist RGC100. RGC100 has a defined chemical structure, with a defined length (100 bp) and molecular weight (64.9 KDa) and a good solubility. RGC100 is stable in serum and activates myeloid DCs through TLR3 targeting, as evidenced by gene silencing experiments. Activation of mouse and human myeloid CD1c(+) DCs by RGC100 leads to secretion of several proinflammatory cytokines. In addition, RGC100 improves the ability of CD1c(+) DCs to stimulate T-cell proliferation. Due to its physicochemical properties and its immunostimulatory properties, RGC100 may represent a promising adjuvant for prophylactic and therapeutic vaccination strategies.

In vitro osteogenic properties of two dental implant surfaces.

Current dental implant research aims at understanding the biological basis for successful implant therapy. The aim of the study was to perform a full characterization of the effect of two commercial titanium (Ti) surfaces, OsseoSpeed and TiOblast, on the behaviour of mouse preosteoblast MC3T3-E1 cells. The effect of these Ti surfaces was compared with tissue culture plastic (TCP). In vitro experiments were performed to evaluate cytotoxicity, cell morphology and proliferation, alkaline phosphatase activity, gene expression, and release of a wide array of osteoblast markers. No differences were observed on cell viability and cell proliferation. However, changes were observed in cell shape after 2 days, with a more branched morphology on OsseoSpeed compared to TiOblast. Moreover, OsseoSpeed surface increased BMP-2 secretion after 2 days, and this was followed by increased IGF-I, BSP, and osterix gene expression and mineralization compared to TiOblast after 14 days. As compared to the gold standard TCP, both Ti surfaces induced higher osteocalcin and OPG release than TCP and differential temporal gene expression of osteogenic markers. The results demonstrate that the gain of using OsseoSpeed surface is an improved osteoblast differentiation and mineralization, without additional effects on cell viability or proliferation.

Clinical color intensity of white spot lesions might be a better predictor of enamel demineralization depth than traditional clinical grading.

INTRODUCTION: The aims of this study were to calculate the volume of white spot lesions by using microcomputed tomography and to determine which clinical attribute of the white spot lesion could better predict its volume: the clinically visible white spot lesion surface area or its color intensity. METHODS: White spot lesions were induced in 8 patients in vivo on 23 healthy premolars destined for extraction during orthodontic treatment by using specially designed plaque-retaining orthodontic bands. After 7 weeks, the premolars were extracted. After extraction, the resulting white spot lesions were photographed and clinically graded. The teeth were analyzed with microcomputed tomography. RESULTS: After 7 weeks, 70% of the teeth developed clinical white spot lesions. Clinically, the size of the lesions varied from minor to severe. Their volumes varied from 0 to 1.2931 mm(3). The traditional grades for white spot lesions correlated significantly with color intensity. A significant correlation was found between white spot lesion color intensity and lesion volume. This correlation was found to be better than that between the white spot lesion clinical score and lesion volume. CONCLUSIONS: Our results indicate that white spot lesion color intensity might predict the depth of enamel demineralization as well as or better than traditional white spot lesion scoring. Therefore, the dentist could use this information when planning treatment for white spot lesions.

EPA covalently bound to smooth titanium surfaces decreases viability and biofilm formation of Staphylococcus epidermidis in vitro.

Colonization of implant surfaces with bacteria should ideally be prevented right from implantation, as bacteria attaching to the surface will form a biofilm, being then well protected against antibiotic treatment. Therefore, implant coatings should combine antibacterial properties with biocompatibility towards their host tissue. We tested a UV-induced covalent coating procedure with eicosapentaenoic acid (EPA) for smooth titanium (Ti) surfaces for its ability to prevent attachment and proliferation of Staphylococcus epidermidis and to allow mineralization of MC3T3-E1 osteoblasts. Bacterial initial attachment was highest for EPA-coated surfaces, but was reduced by vigorous washing, possibly due to low adhesive strength on those surfaces. We found an increase in the ratio of dead bacteria and in overall biofilm after 16 h on Ti surfaces with covalently bound EPA compared to Ti. The UV-induced EPA coating did not impair the ability of MC3T3-E1 preosteoblasts to mineralize, while a reduction in mineralization could be found for UV-irradiated Ti surfaces and UV-irradiated surfaces washed with ethanol compared to Ti. Although in vivo studies are needed to evaluate the clinical significance, our results indicate that covalent coating of Ti surfaces with EPA by UV irradiation decreases the survival of S. epidermidis and maintains the mineralization ability of osteoblasts.

Decreased bone mineral density and reduced bone quality in H(+) /K(+) ATPase beta-subunit deficient mice.

Proton pump inhibitors (PPIs) are widely used against gastroesophageal reflux disease. Recent epidemiological studies suggest that PPI users have an increased risk of fractures, but a causal relationship has been questioned. We have therefore investigated the skeletal phenotype in H(+) /K(+) ATPase beta-subunit knockout (KO) female mice. Skeletal parameters were determined in 6- and 20-month-old KO mice and in wild-type controls (WT). Whole body bone mineral density (BMD) and bone mineral content (BMC) were measured by dual energy X-ray absorptiometry (DXA). Femurs were examined with µCT analyses and break force were examined by a three-point bending test. Plasma levels of gastrin, RANKL, OPG, osteocalcin, leptin, and PTH were analyzed. KO mice had lower whole body BMC at 6 months (0.53 vs. 0.59 g, P = 0.035) and at 20 months (0.49 vs. 0.74 g, P < 0.01) compared to WT as well as lower BMD at 6 months (0.068 vs. 0.072 g/cm(2) , P = 0.026) and 20 months (0.067 vs. 0.077 g/cm(2) , P < 0.01). Mechanical strength was lower in KO mice at the age of 20 months (6.7 vs. 17.9 N, P < 0.01). Cortical thickness at 20 months and trabecular bone volume% at 6 months were significantly reduced in KO mice. Plasma OPG/RANKL ratio and PTH was increased in KO mice compared to controls. H(+) /K(+) ATPase beta subunit KO mice had decreased BMC and BMD, reduced cortical thickness and inferior mechanical bone strength. Whereas the mechanism is uncertain, these findings suggest a causal relationship between long-term PPI use and an increased risk of fractures.

The peroxisome proliferator-activated receptor (PPAR) alpha agonist fenofibrate maintains bone mass, while the PPAR gamma agonist pioglitazone exaggerates bone loss, in ovariectomized rats.

BACKGROUND: Activation of peroxisome proliferator-activated receptor (PPAR)gamma is associated with bone loss and increased fracture risk, while PPARalpha activation seems to have positive skeletal effects. To further explore these effects we have examined the effect of the PPARalpha agonists fenofibrate and Wyeth 14643, and the PPARgamma agonist pioglitazone, on bone mineral density (BMD), bone architecture and biomechanical strength in ovariectomized rats. METHODS: Fifty-five female Sprague-Dawley rats were assigned to five groups. One group was sham-operated and given vehicle (methylcellulose), the other groups were ovariectomized and given vehicle, fenofibrate, Wyeth 14643 and pioglitazone, respectively, daily for four months. Whole body and femoral BMD were measured by dual X-ray absorptiometry (DXA), and biomechanical testing of femurs, and micro-computed tomography (microCT) of the femoral shaft and head, were performed. RESULTS: Whole body and femoral BMD were significantly higher in sham controls and ovariectomized animals given fenofibrate, compared to ovariectomized controls. Ovariectomized rats given Wyeth 14643, maintained whole body BMD at sham levels, while rats on pioglitazone had lower whole body and femoral BMD, impaired bone quality and less mechanical strength compared to sham and ovariectomized controls. In contrast, cortical volume, trabecular bone volume and thickness, and endocortical volume were maintained at sham levels in rats given fenofibrate. CONCLUSIONS: The PPARalpha agonist fenofibrate, and to a lesser extent the PPARaplha agonist Wyeth 14643, maintained BMD and bone architecture at sham levels, while the PPARgamma agonist pioglitazone exaggerated bone loss and negatively affected bone architecture, in ovariectomized rats.

Sinus graft with safescraper: 5-year results.

PURPOSE: In the procedure of sinus floor elevation, autogenous bone, allogenic grafts, and several other bone substitutes are used. However, autogenous bone is still considered the gold standard. Donor sites for autogenous bone are generally the iliac crest, oral cavity, calvarium bone, and tibia. In this work the experience with the use of a Safescraper device for harvesting of autogenous bone is reported and a decision-making algorithm for grafting in sinus floor elevation procedures is proposed. MATERIALS AND METHODS: Forty sinus augmentation procedures were performed in 34 patients. All sinuses were filled with a mixture of autogenous bone and bovine hydroxyapatite. A Safescraper device was used to harvest autologous bone from the maxillary area. Platelet-rich plasma was used to sustain bone placement. Sixty-five dental implants were placed at 4 months with a flapless procedure. A clinical and radiological 5-year retrospective case series of a cohort is reported. RESULTS: In all cases new bone formation was confirmed radiologically and implant placement was performed successfully. Analysis of samples obtained by biopsy with histology and microcomputed tomography showed the presence of mature bone. Healing problems were observed in only 1 case. CONCLUSIONS: Sinus augmentation with bone grafts obtained from oral cavity with a bone scraper device has the advantage of providing autogenous bone without the need for an extra surgical approach. This procedure yields satisfactory results in bone formation, implant survival, and patient satisfaction. When combined with a flapless approach for implant placement, a decrease in the morbidity of the entire process is achieved.

In vivo performance of titanium implants functionalized with eicosapentaenoic acid and UV irradiation.

The aim of this study was to design implant surfaces that attach less to bone but at the same time improve osseous healing for use as temporary bone fracture plates. The strategy was to combine the nonadhesive properties of smooth titanium (Ti) surfaces with the differentiative and anti-inflammatory properties of eicosapentaenoic acid (EPA). Machined Ti implant surfaces coated with a layer of EPA, with or without UV irradiation, were characterized by X-ray photoelectron spectroscopy, and their in vivo performance was evaluated in New Zealand White rabbits. The performance of the functionalised implants was analyzed after 10 weeks of healing by mechanical pull-out testing, molecular biology, and histological and microcomputed tomography analysis. The results indicate that surface functionalization with UV light can reduce bone attachment and volumetric bone mineral density in the peri-implant bone tissue. The presence of EPA on the surfaces enhanced this effect further. Gene expression of bone formation markers showed a trend toward higher mRNA levels in all EPA treated groups. The histological analyses demonstrated lower inflammation in the UV-irradiated group and immature bone formation in all the groups. In conclusion, surface functionalization of Ti implants with UV light and EPA could be a biocompatible coating for reduced bone bonding ability of Ti while promoting bone formation.

Human osteoblastic cells discriminate between 20-kDa amelogenin isoforms.

Enamel matrix derivative (EMD) is used to stimulate healing of alveolar bone after destructive marginal periodontitis; however, the roles of the different EMD constituents are unclear. The aim here was to compare the effect of two EMD fractions (A1 and A2) on primary human osteoblasts cultured in the presence of 50 µg ml(-1) of A1, A2, or EMD. SDS-PAGE showed that A1 and A2 were comprised of amelogenins migrating at around 20 kDa. Fourier transform infrared (FTIR) analysis revealed that A1 and A2 had different secondary structures, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) identified different peptide mass values. Osteoblasts responded differently to A1 and A2. Whereas A1 enhanced the proliferation [measured by the incorporation of 5-bromo-2'-deoxyuridine (BrdU)] of osteoblasts, the expression of runt-related transcription factor-2 (RUNX2) mRNA, and the secretion of interleukin 6 (IL-6) into the cell culture medium, exposure to A2 resulted in increased alkaline phosphatase (ALP) activity, increased expression of CD44 mRNA, and increased secretion of osteoprotegrin (OPG) and receptor activator of nuclear factor-kappaB ligand (RANKL). The level of osteocalcin in the cell culture medium was increased after all treatments, while A2 stimulated the expression of dentin matrix protein 1 (DMP1) mRNA. The results suggest that both A1 and A2 participate in the observed effect of EMD, but have different effects on the expression of osteoblast mRNA and the secretion of osteoblast protein, and thus might facilitate the differentiation of a different phenotype.

A switchable palladium-complexed molecular shuttle and its metastable positional isomers.

We report the design, synthesis, characterization, and operation of a [2]rotaxane in which a palladium-complexed macrocycle can be translocated between 4-dimethylaminopyridine and pyridine monodentate ligand sites via reversible protonation, the metal remaining coordinated to the macrocycle throughout. The substitution pattern of the ligands and the kinetic stability of the Pd-N bond means that changing the chemical state of the thread does not automatically cause a change in the macrocycle's position in the absence of an additional input (heat and/or coordinating solvent/anion). Accordingly, under ambient conditions any of the four sets of protonated and neutral, stable, and metastable co-conformers of the [2]rotaxane can be selected, manipulated, isolated, and characterized.