Combined Therapy of AXL and HDAC Inhibition Reverses Mesenchymal Transition in Diffuse Intrinsic Pontine Glioma.

PURPOSE: Diffuse intrinsic pontine glioma (DIPG) is an incurable type of pediatric brain cancer, which in the majority of cases is driven by mutations in genes encoding histone 3 (H3K27M). We here determined the preclinical therapeutic potential of combined AXL and HDAC inhibition in these tumors to reverse their mesenchymal, therapy-resistant, phenotype. EXPERIMENTAL DESIGN: We used public databases and patient-derived DIPG cells to identify putative drivers of the mesenchymal transition in these tumors. Patient-derived neurospheres, xenografts, and allografts were used to determine the therapeutic potential of combined AXL/HDAC inhibition for the treatment of DIPG. RESULTS: We identified AXL as a therapeutic target and regulator of the mesenchymal transition in DIPG. Combined AXL and HDAC inhibition had a synergistic and selective antitumor effect on H3K27M DIPG cells. Treatment of DIPG cells with the AXL inhibitor BGB324 and the HDAC inhibitor panobinostat resulted in a decreased expression of mesenchymal and stem cell genes. Moreover, this combination treatment decreased expression of DNA damage repair genes in DIPG cells, strongly sensitizing them to radiation. Pharmacokinetic studies showed that BGB324, like panobinostat, crosses the blood-brain barrier. Consequently, treatment of patient-derived DIPG xenograft and murine DIPG allograft-bearing mice with BGB324 and panobinostat resulted in a synergistic antitumor effect and prolonged survival. CONCLUSIONS: Combined inhibition of AXL and HDACs in DIPG cells results in a synergistic antitumor effect by reversing their mesenchymal, stem cell-like, therapy-resistant phenotype. As such, this treatment combination may serve as part of a future multimodal therapeutic strategy for DIPG.

Multiregional Tumor Drug-Uptake Imaging by PET and Microvascular Morphology in End-Stage Diffuse Intrinsic Pontine Glioma.

Inadequate tumor uptake of the vascular endothelial growth factor antibody bevacizumab could explain lack of effect in diffuse intrinsic pontine glioma. Methods: By combining data from a PET imaging study using 89Zr-labeled bevacizumab and an autopsy study, a 1-on-1 analysis of multiregional in vivo and ex vivo 89Zr-bevacizumab uptake, tumor histology, and vascular morphology in a diffuse intrinsic pontine glioma patient was performed. Results: In vivo 89Zr-bevacizumab measurements showed heterogeneity between lesions. Additional ex vivo measurements and immunohistochemistry of cervicomedullary metastasis samples showed uptake to be highest in the area with marked microvascular proliferation. In the primary pontine tumor, all samples showed similar vascular morphology. Other histologic features were similar between the samples studied. Conclusion: In vivo 89Zr-bevacizumab PET serves to identify heterogeneous uptake between tumor lesions, whereas subcentimeter intralesional heterogeneity could be identified only by ex vivo measurements. 89Zr-bevacizumab uptake is enhanced by vascular proliferation, although our results suggest it is not the only determinant of intralesional uptake heterogeneity.

Culture methods of diffuse intrinsic pontine glioma cells determine response to targeted therapies.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive type of brainstem cancer occurring mainly in children, for which there currently is no effective therapy. Current efforts to develop novel therapeutics for this tumor make use of primary cultures of DIPG cells, maintained either as adherent monolayer in serum containing medium, or as neurospheres in serum-free medium. In this manuscript, we demonstrate that the response of DIPG cells to targeted therapies in vitro is mainly determined by the culture conditions. We show that particular culture conditions induce the activation of different receptor tyrosine kinases and signal transduction pathways, as well as major changes in gene expression profiles of DIPG cells in culture. These differences correlate strongly with the observed discrepancies in response to targeted therapies of DIPG cells cultured as either adherent monolayers or neurospheres. With this research, we provide an argument for the concurrent use of both culture conditions to avoid false positive and false negative results due to the chosen method.

Preclinical evaluation of convection-enhanced delivery of liposomal doxorubicin to treat pediatric diffuse intrinsic pontine glioma and thalamic high-grade glioma.

OBJECTIVE Pediatric high-grade gliomas (pHGGs) including diffuse intrinsic pontine gliomas (DIPGs) are primary brain tumors with high mortality and morbidity. Because of their poor brain penetrance, systemic chemotherapy regimens have failed to deliver satisfactory results; however, convection-enhanced delivery (CED) may be an alternative mode of drug delivery. Anthracyclines are potent chemotherapeutics that have been successfully delivered via CED in preclinical supratentorial glioma models. This study aims to assess the potency of anthracyclines against DIPG and pHGG cell lines in vitro and to evaluate the efficacy of CED with anthracyclines in orthotopic pontine and thalamic tumor models. METHODS The sensitivity of primary pHGG cell lines to a range of anthracyclines was tested in vitro. Preclinical CED of free doxorubicin and pegylated liposomal doxorubicin (PLD) to the brainstem and thalamus of naïve nude mice was performed. The maximum tolerated dose (MTD) was determined based on the observation of clinical symptoms, and brains were analyzed after H & E staining. Efficacy of the MTD was tested in adult glioma E98-FM-DIPG and E98-FM-thalamus models and in the HSJD-DIPG-007-Fluc primary DIPG model. RESULTS Both pHGG and DIPG cells were sensitive to anthracyclines in vitro. Doxorubicin was selected for further preclinical evaluation. Convection-enhanced delivery of the MTD of free doxorubicin and PLD in the pons was 0.02 mg/ml, and the dose tolerated in the thalamus was 10 times higher (0.2 mg/ml). Free doxorubicin or PLD via CED was ineffective against E98-FM-DIPG or HSJD-DIPG-007-Fluc in the brainstem; however, when applied in the thalamus, 0.2 mg/ml of PLD slowed down tumor growth and increased survival in a subset of animals with small tumors. CONCLUSIONS Local delivery of doxorubicin to the brainstem causes severe toxicity, even at doxorubicin concentrations that are safe in the thalamus. As a consequence, the authors could not establish a therapeutic window for treating orthotopic brainstem tumors in mice. For tumors in the thalamus, therapeutic concentrations to slow down tumor growth could be reached. These data suggest that anatomical location determines the severity of toxicity after local delivery of therapeutic agents and that caution should be used when translating data from supratentorial CED studies to treat infratentorial tumors.

The effect of different collagen modifications for titanium and titanium nitrite surfaces on functions of gingival fibroblasts.

OBJECTIVES: Targeted modifications of the bulk implant surfaces using bioactive agents provide a promising tool for improvement of the long-term bony and soft tissue integration of dental implants. In this study, we assessed the cellular responses of primary human gingival fibroblasts (HGF) to different surface modifications of titanium (Ti) and titanium nitride (TiN) alloys with type I collagen or cyclic-RGDfK-peptide in order to define a modification improving long-term implants in dental medicine. MATERIALS AND METHODS: Employing Ti and TiN implants, we compared the performance of simple dip coating and anodic immobilization of type I collagen that provided collagen layers of two different thicknesses. HGF were seeded on the different coated implants, and adhesion, proliferation, and gene expression were analyzed. RESULTS: Although there were no strong differences in initial cell adhesion between the groups at 2 and 4 hours, we found that all surface modifications induced higher proliferation rates as compared to the unmodified controls. Consistently, gene expression levels of cell adhesion markers (focal adhesion kinase (FAK), integrin beta1, and vinculin), cell differentiation markers (FGFR1, TGFb-R1), extracellular protein markers (type I collagen, vimentin), and cytoskeletal protein marker aktinin-1 were consistently higher in all surface modification groups at two different time points of investigation as compared to the unmodified controls. CONCLUSION: Our results indicate that simple dip coating of Ti and TiN with collagen is sufficient to induce in vitro cellular responses that are comparable to those of more reliable coating methods like anodic adsorption, chemical cross-linking, or RGD coating. TiN alloys do not possess any positive or adverse effects on HGF. CLINICAL RELEVANCE: Our results demonstrate a simple, yet effective, method for collagen coating on titanium implants to improve the long term integration and stability of dental implants.

Bevacizumab Targeting Diffuse Intrinsic Pontine Glioma: Results of 89Zr-Bevacizumab PET Imaging in Brain Tumor Models.

The role of the VEGF inhibitor bevacizumab in the treatment of diffuse intrinsic pontine glioma (DIPG) is unclear. We aim to study the biodistribution and uptake of zirconium-89 ((89)Zr)-labeled bevacizumab in DIPG mouse models. Human E98-FM, U251-FM glioma cells, and HSJD-DIPG-007-FLUC primary DIPG cells were injected into the subcutis, pons, or striatum of nude mice. Tumor growth was monitored by bioluminescence imaging (BLI) and visualized by MRI. Seventy-two to 96 hours after (89)Zr-bevacizumab injections, mice were imaged by positron emission tomography (PET), and biodistribution was analyzed ex vivo High VEGF expression in human DIPG was confirmed in a publically available mRNA database, but no significant (89)Zr-bevacizumab uptake could be detected in xenografts located in the pons and striatum at an early or late stage of the disease. E98-FM, and to a lesser extent the U251-FM and HSJD-DIPG-007 subcutaneous tumors, showed high accumulation of (89)Zr-bevacizumab. VEGF expression could not be demonstrated in the intracranial tumors by in situ hybridization (ISH) but was clearly present in the perinecrotic regions of subcutaneous E98-FM tumors. The poor uptake of (89)Zr-bevacizumab in xenografts located in the brain suggests that VEGF targeting with bevacizumab has limited efficacy for diffuse infiltrative parts of glial brain tumors in mice. Translating these results to the clinic would imply that treatment with bevacizumab in patients with DIPG is only justified after targeting of VEGF has been demonstrated by (89)Zr-bevacizumab immuno-PET. We aim to confirm this observation in a clinical PET study with patients with DIPG. Mol Cancer Ther; 15(9); 2166-74. ©2016 AACR.

Convection enhanced delivery of carmustine to the murine brainstem: a feasibility study.

BACKGROUND: Systemic delivery of therapeutic agents remains ineffective against diffuse intrinsic pontine glioma (DIPG), possibly due to an intact blood-brain-barrier (BBB) and to dose-limiting toxicity of systemic chemotherapeutic agents. Convection-enhanced delivery (CED) into the brainstem may provide an effective local delivery alternative for DIPG patients. NEW METHOD: The aim of this study is to develop a method to perform CED into the murine brainstem and to test this method using the chemotherapeutic agent carmustine (BiCNU). To this end, a newly designed murine CED catheter was tested in vitro and in vivo. After determination of safety and distribution, mice bearing VUMC-DIPG-3 and E98FM-DIPG brainstem tumors were treated with carmustine dissolved in DW 5% or carmustine dissolved in 10% ethanol. RESULTS: Our results show that CED into the murine brainstem is feasible and well tolerated by mice with and without brainstem tumors. CED of carmustine dissolved in 5% DW increased median survival of mice with VUMC-DIPG-3 and E98FM-DIPG tumors with 35% and 25% respectively. Dissolving carmustine in 10% ethanol further improved survival to 45% in mice with E98FM-DIPG tumors. COMPARISON WITH EXISTING METHODS: Since genetically engineered and primary DIPG models are currently only available in mice, murine CED studies have clear advantages over CED studies in other animals. CONCLUSION: CED in the murine brainstem can be performed safely, is well tolerated and can be used to study efficacy of chemotherapeutic agents orthotopically. These results set the foundation for more CED studies in murine DIPG models.

Human pontine glioma cells can induce murine tumors.

Diffuse intrinsic pontine glioma (DIPG), with a median survival of only 9 months, is the leading cause of pediatric brain cancer mortality. Dearth of tumor tissue for research has limited progress in this disease until recently. New experimental models for DIPG research are now emerging. To develop preclinical models of DIPG, two different methods were adopted: cells obtained at autopsy (1) were directly xenografted orthotopically into the pons of immunodeficient mice without an intervening cell culture step or (2) were first cultured in vitro and, upon successful expansion, injected in vivo. Both strategies resulted in pontine tumors histopathologically similar to the original human DIPG tumors. However, following the direct transplantation method all tumors proved to be composed of murine and not of human cells. This is in contrast to the indirect method that included initial in vitro culture and resulted in xenografts comprising human cells. Of note, direct injection of cells obtained postmortem from the pons and frontal lobe of human brains not affected by cancer did not give rise to neoplasms. The murine pontine tumors exhibited an immunophenotype similar to human DIPG, but were also positive for microglia/macrophage markers, such as CD45, CD68 and CD11b. Serial orthotopic injection of these murine cells results in lethal tumors in recipient mice. Direct injection of human DIPG cells in vivo can give rise to malignant murine tumors. This represents an important caveat for xenotransplantation models of DIPG. In contrast, an initial in vitro culture step can allow establishment of human orthotopic xenografts. The mechanism underlying this phenomenon observed with direct xenotransplantation remains an open question.

Therapeutic hypercapnia prevents bleomycin-induced pulmonary hypertension in neonatal rats by limiting macrophage-derived tumor necrosis factor-α.

Bleomycin-induced lung injury is characterized in the neonatal rat by inflammation, arrested lung growth, and pulmonary hypertension (PHT), as observed in human infants with severe bronchopulmonary dysplasia. Inhalation of CO(2) (therapeutic hypercapnia) has been described to limit cytokine production and to have anti-inflammatory effects on the injured lung; we therefore hypothesized that therapeutic hypercapnia would prevent bleomycin-induced lung injury. Spontaneously breathing rat pups were treated with bleomycin (1 mg/kg/d ip) or saline vehicle from postnatal days 1-14 while being continuously exposed to 5% CO(2) (Pa(CO(2)) elevated by 15-20 mmHg), 7% CO(2) (Pa(CO(2)) elevated by 35 mmHg), or normocapnia. Bleomycin-treated animals exposed to 7%, but not 5%, CO(2), had significantly attenuated lung tissue macrophage influx and PHT, as evidenced by normalized pulmonary vascular resistance and right ventricular systolic function, decreased right ventricular hypertrophy, and attenuated remodeling of pulmonary resistance arteries. The level of CO(2) neither prevented increased tissue neutrophil influx nor led to improvements in decreased lung weight, septal thinning, impaired alveolarization, or decreased numbers of peripheral arteries. Bleomycin led to increased expression and content of lung tumor necrosis factor (TNF)-α, which was found to colocalize with tissue macrophages and to be attenuated by exposure to 7% CO(2). Inhibition of TNF-α signaling with the soluble TNF-2 receptor etanercept (0.4 mg/kg ip from days 1-14 on alternate days) prevented bleomycin-induced PHT without decreasing tissue macrophages and, similar to CO(2), had no effect on arrested alveolar development. Our findings are consistent with a preventive effect of therapeutic hypercapnia with 7% CO(2) on bleomycin-induced PHT via attenuation of macrophage-derived TNF-α. Neither tissue macrophages nor TNF-α appeared to contribute to arrested lung development induced by bleomycin. That 7% CO(2) normalized pulmonary vascular resistance and right ventricular function without improving inhibited airway and vascular development suggests that vascular hypoplasia does not contribute significantly to functional changes of PHT in this model.

Cyclo-DfKRG peptide modulates in vitro and in vivo behavior of human osteoprogenitor cells on titanium alloys.

The first aim of the present study was to investigate the capacity of a cyclo-DfKRG-coated hydroxyapatite-titanium alloy (Ti-HA-RGD) to activate in vitro human osteoprogenitor cells adhesion and differentiation. The second purpose was to examine in vivo the role of a autologous cell seeding on cyclo-DfKRG-functionalized materials to provide bone repair after implantation in femoral condyle of rabbits. Our in vitro results have demonstrated that both titanium alloy functionalized with hydroxyapatite (Ti-HA-RGD and Ti-HA) contributed to higher cell adhesion than titanium alloy alone respectively 85 and 55% vs 15% compared to tissue culture polystyrene after one hour of cell seeding. As for differentiation, after 3 days of culture, Ti-HA presented the highest increase of ALP mRNA of all surfaces studied. Ti-HA-RGD showed an intermediate value about half as high as Ti-HA. Moreover after 3 days, both Ti-HA and Ti-HA-RGD surfaces showed the highest increase of cbfa1 mRNA expression. Two weeks following implantation, in vivo findings revealed that percentage of lacunae contact observed with pre-cellularized Ti-HA-RGD samples remains significantly lower than with Ti-HA group (10.5+/-9.6 % vs 33.7+/-11.5 %, P<0.03). Meanwhile, RGD peptide coating had no significant additional effect on the bone implant contact and area. Moreover, histomorphometry analysis revealed that implantation of pre-cellularized RGD coated materials with ROP cells increased significantly peri-implant fibrous area (24+/-11.6% vs 3+/-1.7% for Ti-HA-RGD, P<0.02). RGD coatings demonstrated osteoblastic adhesion, differentiation and in vivo bone regeneration at most equivalent to HA coatings.

Effect of modifications of dual acid-etched implant surfaces on peri-implant bone formation. Part I: organic coatings.

OBJECTIVE: The aim of the present study was to test the hypothesis that peri-implant bone formation can be improved by modifying dual acid-etched (DAE) implant surfaces using organic coatings that enhance cell adhesion and osteogenic differentiation. MATERIAL AND METHODS: Ten adult female foxhounds received experimental titanium implants in the mandible 3 months after removal of all premolar teeth. Six types of implants were evaluated in each animal: (i) implants with a machined surface (MS), (ii) implants with a DAE surface topography, (iii) implants with an acid-etched surface coated with RGD peptides, (iv) implants with an acid-etched surface coated with collagen I, (v) implants with an acid-etched surface coated with collagen I and chondroitin sulphate (CS), (vi) implants with an acid-etched surface coated with collagen I and CS and recombinant human bone morphogenetic protein-2. Peri-implant bone regeneration was assessed by histomorphometry after 1 and 3 months in five dogs each by measuring bone implant contact (BIC) and the bone volume density (BVD) of the newly formed peri-implant bone. RESULTS: After 1 month, mean BIC was significantly higher in the coated implants group than in the MS group. There was no significant difference when mean BIC in the DAE group was compared with implants with any of the organic coatings, but the difference was significant when compared with the MS implants. Differences in mean BVD value did not reach significance between any of the surfaces. After 3 months, the same held true for the mean BIC of all the groups except for Coll I. Mean volume density of the newly formed bone was higher in all the surface modifications, albeit without statistical significance. CONCLUSIONS: It is concluded that with the exception of Coll I, the tested organic surface coatings on DAE surfaces did not improve peri-implant bone formation when compared with the DAE surfaces but enhanced BIC when compared with the MSs.

Effect of modifications of dual acid-etched implant surfaces on periimplant bone formation. Part II: calcium phosphate coatings.

The aim of the present study was to test the hypothesis that calcium phosphate coatings of dual acid-etched surfaces (DAEs) can improve periimplant bone regeneration. Ten adult female foxhounds received experimental titanium screw implants in the mandible 3 months after removal of all premolar teeth. Five types of surface states were evaluated in each animal: (i) implants with a machined surface (MS) (Control 1); (ii) implants with a DAE (Control 2); (iii) implants with a DAE coated with collagen I (Control 3); (iv) implants with a DAE with mineralized collagen I; and (v) implants with a DAE with a hydroxylapatite (HA) coating. Periimplant bone regeneration was assessed by histomorphometry after 1 and 3 months in five dogs each by measuring bone implant contact (BIC) and the volume density of the newly formed periimplant bone (BVD). After 1 month, mean BIC of experimental implants did not differ significantly from implants with DAE and collagen-coated surfaces, but was significantly higher than the MS implants. BVD was enhanced significantly only in implants with mineralized collagen coating compared with DAE and collagen-coated controls. After 3 months, the mean values of BIC had increased significantly in the group of implants with HA and mineralized collagen coating but were not significantly different from implants with DAE and collagen-coated surfaces. The same held true for the mean BVD values. In conclusion, the present study could not verify the hypothesis that calcium phosphate coatings of DAEs in the present form enhanced periimplant bone formation compared with the DAE surface alone.

Feasibility study using surface-enhanced Raman spectroscopy for the quantitative detection of tyrosine and serine phosphorylation.

We investigate the feasibility of colloid-based surface enhanced Raman scattering (SERS) as a highly sensitive technique for detecting peptide phosphorylation at serine and tyrosine residues. Using the recently reported drop-coating deposition Raman method we validate our SERS spectra against normal Raman spectra that would otherwise be unobtainable at such low concentrations. Compared with existing techniques for quantifying peptide phosphorylation, such as high-performance liquid chromatography (HPLC), the short scanning and processing time associated with SERS makes it an attractive alternative for near-real-time measurement at sub micro-molar concentrations. Following pre-processing by Savistky-Golay second derivative (SGSD), the degree of phosphorylation of synthetic peptides is determined using multivariate spectral classification, interval partial least squares (iPLS). Furthermore, our results show that the technique is robust to interference from complex proteins and other phosphorylated compounds present at concentrations typically found in a screening assay.

Resonance frequency measurements of implant stability in the dog mandible: experimental comparison with histomorphometric data.

The aim of the present study was to test the hypothesis that measurements of implant stability using resonance frequency analysis (RFA) correlate with histomorphometric data of bone anchorage. Ten adult female foxhounds received a total of 80 implants in their mandibles 3 months after removal of all premolar teeth. At the time of implant placement, torque required for bone tapping was registered as a measure of bone density and immediately after placement implant stability was assessed using RFA. RFA measurements were repeated at the time of implant retrieval after 1 month (5 dogs) and 3 months (5 dogs). Peri-implant bone regeneration was assessed histomorphometrically by measuring bone-implant contact (BIC) and the volume density of the newly formed peri-implant bone (BVD). RFA values at the time of implant placement did not correlate with the torque required to tap the bone for implant placement. After 1 and 3 months, RFA values were significantly increased compared with baseline values. BIC and BVD, however, had increased significantly during this interval. There was no correlation between bone-implant contact and RFA values nor between peri-implant bone density and RFA values. Thus, the hypothesis could not be verified. It is concluded that the validity of the individual measurement of implant stability using RFA should be considered with caution.

High-throughput lead finding and optimisation for GPCR targets.

Driven by past successes and the detailed knowledge of signalling cascades and physiological processes, G-protein-coupled receptors are taking a prominent place in the portfolios of many pharmaceutical companies. To successfully address this target class, scientists need not only a good understanding of the specific receptor under investigation, but also the right tools from assay technology, reagent production to a hit-to-lead process that acknowledges the importance of parameters beyond potency and embraces the gain in knowledge of the last decade. This manuscripts attempts to summarise some of the changes and progress made across the pharmaceutical industry to design an efficient and effective strategy for finding and optimising small molecules modulating the activity of GPCRs.

G-protein coupled receptor assays: to measure affinity or efficacy that is the question.

Cell-based assays have always played an important role in the pharmaceutical industry, providing information about the functional effects of compounds. These functional assays have traditionally accompanied facile biochemical high throughput screening programmes, being applied as secondary assays in the later stages of lead development. However, with the disappointing reality that there is not likely to be a plethora of novel, druggable targets in the post-genomic era, the role of cell-based assays in drug discovery is beginning to change. Competition to develop the "best" agents for well established targets and find more effective ways of identifying "novel" agents is driving the industry towards a "quality" versus "quantity" approach. Advances in genetic engineering, automation compatible functional assay technologies and the introduction of more sophisticated robotic systems, have facilitated the application of cell-based assays to primary screening. However, despite some apparent success to move these assays into the routine "toolbox" for high throughput screening, certain preconceptions and concerns about cell-based assays persist and the subject remains a topic of much debate. Here we use examples from the screening portfolio at Pfizer, Sandwich, to discuss the practical and theoretical considerations of employing cell-based assays in HTS with a focus on G-protein coupled receptors.

Functionalization of dental implant surfaces using adhesion molecules.

The aim of the present study was to test the hypothesis that organic coating of titanium screw implants that provides binding sites for integrin receptors can enhance periimplant bone formation. Ten adult female foxhounds received experimental titanium screw implants in the mandible 3 months after removal of all premolar teeth. Four types of implants were evaluated in each animal: (1) implants with machined titanium surface, (2) implants coated with collagen I, (3) implants with collagen I and cyclic RGD peptide coating (Arg-Gly-Asp) with low RGD concentrations (100 micromol/mL), and (4) implants with collagen I and RGD coating with high RGD concentrations (1000 micromol/mL). Periimplant bone regeneration was assessed histomorphometrically after 1 and 3 months in five dogs each by measuring bone implant contact (BIC) and the volume density of the newly formed periimplant bone (BVD). After 1 month, BIC was significantly enhanced only in the group of implants coated with the higher concentration of RGD peptides (p = 0.026). Volume density of the newly formed periimplant bone was significantly higher in all implants with organic coating. No significant difference was found between collagen coating and RGD coatings. After 3 months, BIC was significantly higher in all implants with organic coating than in implants with machined surfaces. Periimplant BVD was significantly increased in all coated implants in comparison to machined surfaces also. It was concluded that organic coating of machined screw implant surfaces providing binding sites for integrin receptors can enhance bone implant contact and periimplant bone formation.

Electrochemically assisted deposition of thin calcium phosphate coatings at near-physiological pH and temperature.

An electrochemical method for the deposition of calcium phosphate phases on titanium surfaces using the galvanostatic mode is presented. Deposition was performed in a (Ca(2+) / H(x)PO(4) ((3-x)-))-containing electrolyte near physiological conditions with regard to pH (6.4) and temperature (36 degrees C). Cathodic alkalization leads first to the formation of a thin homogeneous layer that shows a nanoscale surface topography of alternating wall-like elevations and channels. It is thought that these channels in the calcium phosphate prelayer are formed as pathways for hydroxyl ions and hydrogen. Upon this layer, spheres of amorphous calcium phosphate (ACP) are formed as indicated by Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy. According to transmission electron microscopy images, these spheres consist of small clusters of calcium phosphate (approximately 30 nm) and can grow up to 300 nm in diameter. Characteristic for this ACP is a high water content as seen by FTIR. As a function of current density, the ACP is then transformed into crystalline hydroxyapatite (HAP), which was identified using FTIR and X-ray diffraction. The morphology of the HAP crystals can be described as needles with dimensions of <500-nm length and <60-nm width. By choice of different electrochemical parameters, a homogeneous coating of either ACP, HAP, or the intermediate phase can be achieved, as shown in a kinetic phase diagram, thus allowing the formation of coatings with different properties in solubility and morphology.

Biological performance of biomimetic calcium phosphate coating of titanium implants in the dog mandible.

The aim of the present study was to analyze the in vivo effect of biomimetic calcium phosphate coating of titanium implants on periimplant bone formation and bone-/implant contact. Five types of implants were used: 1) Ti6Al4V implants with a polished surface; 2) Ti6Al4V implants with collagen coating; 3) Ti6Al4V implants with a mineralized collagen layer; 4) Ti6Al4V implants with sequential coating of hydroxyapatite (HA) and collagen; and 5) Ti6Al4V implants with HA coating only. All implants had square cross sections with an oblique diameter of 4.6 mm and were inserted press fit into trephine burr holes of 4.6 mm in the mandibles of ten beagle dogs. The implants of five animals each were evaluated after a healing period of 1 month and 3 months, respectively, during which time sequential fluorochrome labeling of bone formation had been performed. Bone formation was evaluated by morphometric measurement of the newly formed bone around the implants and the percentage of implant bone contact. After 1 month, there was a significantly higher percentage of mean bone/implant contact in the HA-coated implants compared to those with polished surface and those with the collagen-coated surface. After 3 months, these differences were not present anymore. Bone apposition was significantly higher next to implants with sequential HA/collagen coating compared to polished surfaces and mineralized collagen layer. It is concluded that biomimetic coating of titanium implants with HA has shown the clearest trend to increase bone-implant contact in the early ingrowth period. The addition of collagen to an HA coating layer may hold some promise when used as sequential HA/collagen coating with mineralized collagen as the surface layer.

Biomimetic coatings functionalized with adhesion peptides for dental implants.

A complete biological integration into the surrounding tissues (bone, gingiva) is a critical step for clinical success of a dental implant. In this work biomimetic coatings consisting either of collagen type I (for the gingiva region) and hydroxyapatite (HAP) or mineralized collagen (for the bone interface) have been developed as suitable surfaces regarding the interfaces. Additionally, using these biomimetic coatings as a matrix, adhesion peptides were bound to further increase the specificity of titanium implant surfaces. To enhance cell attachment in the gingiva region, a linear adhesion peptide developed from a laminin sequence (TWYKIAFQRNRK) was bound to collagen, whereas for the bone interface, a cyclic RGD peptide was bound to HAP and mineralized collagen using adequate anchor systems. The biological potential of these coatings deduced from cell attachment experiments with HaCaT human keratinocytes and MC3T3-E1 mouse osteoblasts showed the best results for collagen and laminin sequence coating for the gingiva region and mineralized collagen and RGD peptide coatings for regions with bone contact. Our concept opens promising approaches to improve the biological integration of dental implants.