Osteoimmune regulation underlies oral implant osseointegration and its perturbation.

In the field of biomaterials, an endosseous implant is now recognized as an osteoimmunomodulatory but not bioinert biomaterial. Scientific advances in bone cell biology and in immunology have revealed a close relationship between the bone and immune systems resulting in a field of science called osteoimmunology. These discoveries have allowed for a novel interpretation of osseointegration as representing an osteoimmune reaction rather than a classic bone healing response, in which the activation state of macrophages ((M1-M2 polarization) appears to play a critical role. Through this viewpoint, the immune system is responsible for isolating the implant biomaterial foreign body by forming bone around the oral implant effectively shielding off the implant from the host bone system, i.e. osseointegration becomes a continuous and dynamic host defense reaction. At the same time, this has led to the proposal of a new model of osseointegration, the foreign body equilibrium (FBE). In addition, as an oral wound, the soft tissues are involved with all their innate immune characteristics. When implant integration is viewed as an osteoimmune reaction, this has implications for how marginal bone is regulated. For example, while bacteria are constitutive components of the soft tissue sulcus, if the inflammatory front and immune reaction is at some distance from the marginal bone, an equilibrium is established. If however, this inflammation approaches the marginal bone, an immune osteoclastic reaction occurs and marginal bone is removed. A number of clinical scenarios can be envisioned whereby the osteoimmune equilibrium is disturbed and marginal bone loss occurs, such as complications of aseptic nature and the synergistic activation of pro-inflammatory pathways (implant/wear debris, DAMPs, and PAMPs). Understanding that an implant is a foreign body and that the host reacts osteoimmunologically to shield off the implant allows for a distinction to be drawn between osteoimmunological conditions and peri-implant bone loss. This review will examine dental implant placement as an osteoimmune reaction and its implications for marginal bone loss.

Insulin released from titanium discs with insulin coatings-Kinetics and biological activity.

Local administration of insulin from a titanium surface has been demonstrated to increase bone formation in non-diabetic rats. The authors hypothesized that insulin was released from the titanium surface and with preserved biological activity after the release. Thus, in the present in vitro study, human recombinant insulin was immobilized onto titanium discs, and the insulin release kinetics was evaluated using Electro-chemiluminescence immunoassay. Neutral Red uptake assay and mineralization assay were used to evaluate the biological effects of the released insulin on human osteoblast-like MG-63 cells. The results confirmed that insulin was released from titanium surfaces during a six-week period. Etching the disc prior to insulin coating, thickening of the insulin coating and incubation of the discs in serum-enriched cell culture medium increased the release. However, longer storage time decreased the release of insulin. Furthermore, the released insulin had retained its biological activity, as demonstrated by the significant increase in cell number and a stimulated mineralization process, upon exposure to released insulin. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1847-1854, 2017.

Hydroxyapatite coating affects the Wnt signaling pathway during peri-implant healing in vivo.

Owing to its bio- and osteoconductivity, hydroxyapatite (HA) is a widely used implant material, but its osteogenic properties are only partly evaluated in vitro and in vivo. The present study focused on bone healing adjacent to HA-coated titanium (Ti) implants, with or without incorporated lithium ions (Li(+)). Special attention was given to the Wnt signaling pathway. The implants were inserted into rat tibia for 7 or 28 days and analyzed ex vivo, mainly by histomorphometry and quantitative real-time polymerase chain reaction (qPCR). HA-coated implants showed, irrespective of Li(+) content, bone-implant contact (BIC) and removal torque values significantly higher than those of reference Ti. Further, the expression of OCN, CTSK, COL1A1, LRP5/6 and WISP1 was significantly higher in implant-adherent cells of HA-coated implants, with or without Li(+). Significantly higher ß-catenin expression and significantly lower COL2A1 expression were observed in peri-implant bone cells from HA with 14 ng cm(-2) released Li(+). Interestingly, Ti implants showed a significantly larger bone area (BA) in the threads than HA with 39 ng cm(-2) released Li(+), but had a lower BIC than any HA-coated implant. This study shows that HA, with or without Li(+), is a strong activator of the Wnt signaling pathway, and may to some degree explain its high bone induction capacity.

Bone response to surface-modified titanium implants: studies on the early tissue response to implants with different surface characteristics.

In a series of experimental studies, the bone formation around systematically modified titanium implants is analyzed. In the present study, three different surface modifications were prepared and evaluated. Glow-discharge cleaning and oxidizing resulted in a highly stoichiometric TiO2 surface, while a glow-discharge treatment in nitrogen gas resulted in implants with essentially a surface of titanium nitride, covered with a very thin titanium oxide. Finally, hydrogen peroxide treatment of implants resulted in an almost stoichiometric TiO2, rich in hydroxyl groups on the surface. Machined commercially pure titanium implants served as controls. Scanning Auger Electron Spectroscopy, Scanning Electron Microscopy, and Atomic Force Microscopy revealed no significant differences in oxide thickness or surface roughness parameters, but differences in the surface chemical composition and apparent topography were observed. After surface preparation, the implants were inserted in cortical bone of rabbits and evaluated after 1, 3, and 6 weeks. Light microscopic evaluation of the tissue response showed that all implants were in contact with bone and had a large proportion of newly formed bone within the threads after 6 weeks. There were no morphological differences between the four groups. Our study shows that a high degree of bone contact and bone formation can be achieved with titanium implants of different surface composition and topography.

Raloxifene and alendronate containing thin mesoporous titanium oxide films improve implant fixation to bone.

This study tested the hypothesis that osteoporosis drug-loaded mesoporous TiO2 implant coatings can be used to improve bone-implant integration. Two osteoporosis drugs, Alendronate (ALN) and Raloxifene (RLX), were immobilized in nanoporous oxide films prepared on Ti screws and evaluated in vivo in rat tibia. The drug release kinetics were monitored in vitro by quartz crystal microbalance with dissipation and showed sustained release of both drugs. The osteogenic response after 28days of implantation was evaluated by quantitative polymerase chain reaction (qPCR), removal torque, histomorphometry and ultrastructural interface analysis. The drug-loaded implants showed significantly improved bone fixation. In the case of RLX, stronger bone-remodelling activity was observed compared with controls and ALN-loaded implants. The ultrastructural interface analysis revealed enhanced apatite formation inside the RLX coating and increased bone density outside the ALN coating. Thus, this novel combination of a thin mesoporous TiO2 carrier matrix and appropriate drugs can be used to accelerate implant fixation in trabecular bone.

Effects of locally administered insulin on bone formation in non-diabetic rats.

The possibility to control bone formation would be favorable in many areas of medicine, where bone defects is still a major challenge. Insulin has been suggested to exert both systemic and local anabolic effects in bone tissues. This raised the question whether locally administrated insulin could provide new therapeutic strategies for patients with local bone defects and impaired bone healing. The aim of this study was to evaluate bone formation in non-diabetic rats when local insulin is administered. This study differs from previous reports in two aspects: the use of non-diabetic animals and locally administered insulin. Twenty-four implants were inserted into 12 rats-one insulin-coated and one control-in each tibia for four weeks. Interferometry and histomorphometry were used to evaluate the surface topography and bone formation, respectively. Results demonstrated no significant changes in surface topography after insulin immobilization. Histomorphometry revealed significantly more bone around the insulin-coated implants (BA) (p = 0.005) and a similar amount of bone at the implant surface (BIC) (p = 0.117) compared with the controls. It was concluded that locally administered insulin from a titanium implant surface has the potential to increase bone formation not only in diabetic subjects but also in non-diabetic subjects.

Healing of complement activating Ti implants compared with non-activating Ti in rat tibia.

Recent studies have revealed that ozone ultraviolet (UVO) illumination of titanium (Ti) implants improves bone-implant anchorage by altering the physico-chemical and immune activating properties of the titanium dioxide (TiO(2)) layer. In the present rat tibia model, the authors compared the early events of inflammation and bone formation around UVO-treated Ti and complement activating immunoglobin g (IgG)-coated Ti. Machined Ti and machined Ti coated with a physical vapour-deposited Ti layer were used as references. Screw-shaped test and reference implants were implanted into rat tibia and harvested after 1, 7 and 28 days. Messenger RNA expression of implant adhered cells and peri-implant tissue ~250 µm from the surface were subsequently analysed with regard to IL-1ß, TNF-α, osteocalcin, cathepsin K, BMP-2 and PDGF. Separate implants were retrieved after 7 and 28 days for removal torque measurements, and histological staining and histomorphometric analysis of bone area and bone-to-implant contact. While enhanced expression of inflammatory markers, TNF-α and IL-1ß, was observed on IgG-coated surfaces throughout the observation time, UVO-treated surfaces indicated a significantly lower early inflammatory response. In the early phases (1 and 7 days), the UVO-treated surfaces displayed a significantly higher expression of osteoblast markers BMP-2 and osteocalcin. In summary, complement activating Ti implants elicited a stronger inflammatory response than UVO-treated Ti, with low complement activation during the first week of healing. In spite of this, the UVO-treated Ti induced only marginally more bone growth outside the implants.

Local bisphosphonate release versus hydroxyapatite coating for stainless steel screw fixation in rat tibiae.

Implant fixation in bone can be improved by a coating that delivers bisphosphonates locally, or by a hydroxyapatite (HA) coating. In this study, we compared these different types of coatings. For mechanical testing, 30 rats were assigned into three groups, and similar screws were implanted bilaterally in the proximal tibiae. The rats received screws that were either uncoated, coated with nano-crystalline hydroxyapatite or coated with a bisphosphonate releasing protein matrix. After 4 weeks, one screw was subjected to pull-out testing, and the contra-lateral one to torsion testing. For morphology, 30 rats were assigned to similar treatment groups, but received only one screw each. Bisphosphonates enhanced the pull-out force by 41% (P = 0.02) compared to controls, HA increased the pull-out force although not significantly. Conversely, HA increased the maximal torque by 64% (P = 0.02). Morphometry showed higher bone volume around bisphosphonate screws in comparison to HA-coated screws (P < 0.001) and controls (P < 0.001). The results suggest that bisphosphonates improve fixation by increasing the amount of surrounding bone, whereas HA mainly improves bone to implant attachment.

Protein adsorption on thin films of carbon and carbon nitride monitored with in situ ellipsometry.

Thin films of amorphous carbon and amorphous, graphitic and fullerene-like carbon nitride were deposited by reactive magnetron sputtering and optically characterized with spectroscopic ellipsometry. Complementary studies using scanning electron microscopy and atomic force microscopy were performed. The films were exposed to human serum albumin (HSA) and the adsorption was monitored in situ using dynamic ellipsometry. From the ellipsometric data the adsorbed amount of proteins was quantified in terms of surface mass density using de Feijter's model. The results indicate larger adsorption of proteins onto the amorphous films compared to the films with a more textured structure. Complementary studies with 125I-labeled HSA showed an apparent protein adsorption up to six times larger compared to the ellipsometry measurement. In addition, the four types of films were incubated in blood plasma followed by exposure to anti-fibrinogen, anti-HMWK or anti-C3c, revealing the materials' response to complement and contact activation. The amorphous and graphitic carbon nitride exhibit rather high immune activity compared to a titanium reference, whereas the amorphous carbon and the fullerene-like CNx show less immune complement deposition. Compared to the reference, all films exhibit indications of a stronger ability to initiate the intrinsic pathway of coagulation. Finally, the surfaces' bone-bonding ability was investigated by examination of their ability to form calcium phosphate crystals in a simulated body fluid, with a-CNx depositing most calcium phosphate after 21 days of incubation.

Bisphosphonate coating might improve fixation of dental implants in the maxilla: a pilot study.

This pilot study evaluates the clinical stability of bisphosphonate-coated dental implants placed using a two-stage surgical procedure in five patients. Each patient received seven regular Brånemark implants, one of which was coated with bisphosphonate in a fibrinogen matrix. The coated implant was inserted where the bone was expected to have the least favourable quality. The level of the marginal bone around each implant was measured by intraoral periapical radiographs and implant stability was recorded using resonance frequency measurements. Frequency values (ISQ) were obtained peroperatively before flap closure and after 6 months at abutment connection. At abutment connection the bisphosphonate-coated implants were removed en bloc in two patients for histological examination. An animal experiment had previously confirmed that gamma-sterilization did not reduce bioactivity of the bisphosphonate coating. In each patient, the bisphosphonate-coated implant showed the largest improvement in ISQ level of all implants. Their values at the start tended to be lower, and the absolute value at 6 months did not differ. No complications occurred with the coated implants. Histology showed no abnormalities. Improvement in ISQ values was an expected effect of the bisphosphonate coating, but could be due to the choice of insertion site. This finding warrants a randomized, blinded study.

Formation and cross-linking of fibrinogen layers monitored with in situ spectroscopic ellipsometry.

Thick matrices of fibrinogen with incorporation of a matrix metalloproteinase inhibitor were covalently bonded on functionalized silicon surfaces using an ethyl-3-dimethyl-aminopropyl-carbodiimide and N-hydroxy-succinimide affinity ligand coupling chemistry. The growth of the structure was followed in situ using dynamic ellipsometry and characterized at steady-state with spectroscopic ellipsometry. The growth was compared with earlier work on ex situ growth of fibrinogen layers studied by single wavelength ellipsometry. It is found that in situ growth and ex situ growth yield different structural properties of the formed protein matrix. Fibrinogen matrices with thicknesses up to 58 nm and surface mass densities of 1.6 microg/cm2 have been produced.

Stainless steel screws coated with bisphosphonates gave stronger fixation and more surrounding bone. Histomorphometry in rats.

Coating of stainless steel screws with bisphosphonate in a fibrinogen matrix leads to an enhancement of the pullout strength 2 weeks after insertion in rat tibiae. This effect then increases over time until at least 8 weeks. The pullout force reflects the mechanical properties of the bone within the threads, which acts as a screw nut. The aim of the present study was to find descriptive and morphometric histological correlates to the increased pullout strength. Because the bisphosphonates are applied via the implant surface, we also measured bone to implant contact and how far away from the surface any effects could be seen. Stainless steel screws underwent one of three treatments: uncoated control, controls coated with a layer of cross-linked fibrinogen, or screws further modified with bisphosphonates covalently linked and physically adsorbed to the fibrinogen layer. At 1 (n=33) and 8 (n=27) weeks, bone to implant contact and bone area density in the threads were measured, as well as bone area density at 250 and 500 microm from the outer edge of the threads. Additionally, removal torque for each screw treatment was measured at 2 weeks (n=28). At 8 weeks, the part of the bisphosphonate screw that was located in the marrow cavity had become surrounded with bone, whereas there was almost no bone surrounding the controls. The bone area density in the threads along the entire bisphosphonate screw was increased by 40% compared with uncoated controls, and at 250 microm distance it was more than doubled. At 1 week, coated screws had less implant-bone contact, but at 8 weeks there was no difference between uncoated and bisphosphonate-coated screws. The bisphosphonate screws had 50% increased removal torque at 2 weeks compared to uncoated screws. Howship's lacunae and osteoclasts were found near the screws with bisphosphonates at 8 weeks, suggesting that some bone remodeling took place near the implant, in spite of the presence of bisphosphonates.

Detection of surface bound complement at increasing serum anticoagulant concentrations.

Surface mediated immune complement activation can be detected by a variety of antibody utilizing methods such as ELISA, fluorescence- or radiolabelling techniques, QCM, and ellipsometry. In the present work we investigated how the common anticoagulants heparin, dalteparin, fondaparinux and sodium citrate affected the binding of anti-complement factor 3c (anti-C3c) on a model complement activator surface, immobilised IgG, after incubation in human blood serum. The results show, as expected, that different anticoagulants affect the antibody binding differently. Increasing amounts of heparin, dalteparin and sodium citrate in normal serum resulted in a decreasing anti-C3c binding. The antibody deposition was not sensitive for the fondaparinux concentration. Surprisingly high concentrations of anti-coagulantia were needed to completely eradicate the antibody binding. Experiments in EGTA-serum showed that anticoagulants interfered directly with both the classical and alternative pathways. Control C3a-des arg ELISA measurements show that the lowered antibody surface binding was not a result of complement depletion in serum. Kallikrein generation by hydrophilic glass surfaces was not affected by high anticoagulant concentrations.

Inflammatory response to titanium surfaces with fibrinogen and catalase coatings: an in vitro study.

The aim of the present study was to evaluate the possibility to modulate the early inflammatory response in vitro by coating titanium surfaces with candidate proinflammatory (fibrinogen coated turned titanium "Fib") and antiinflammatory proteins (catalase on top of fibrinogen coated turned titanium "Cat"). Additionally, turned titanium surfaces (Ti) were used as controls. The discs were incubated with human mononuclear cells. Adhered cells were investigated with respect to number, viability, differentiation (acute marker 27E10 vs. chronic marker RM3/1), and cytokine production (TNF-alpha and IL-10), after 24 and 72 h. The results indicated that it is possible to modulate the inflammatory response with protein coatings. However, the strongest inflammatory response, indicated by increased number of adhered cells and release of pro and antiinflammatory mediators, was induced by Cat. Furthermore, the cytokine production on this surface was not sensitive to LPS stimulation. Differentiation measured as the expression of the chronic cell surface marker, dominated after 72 h for all surface modifications and Cat displayed an increased number compared to the others. A decrease in the total number of adhered cells and amounts of TNF-alpha were observed on all surfaces over time. The cell viability was, in general, high for all tested surfaces. In conclusion, the study proved it possible to influence the early inflammatory response in vitro by immobilizing protein coatings to titanium surfaces. However, the catalase surface demonstrated the strongest inflammatory response, and the possibility to selectively use the potent antiinflammatory capacity of catalase needs to be further evaluated.

Surface immobilized bisphosphonate improves stainless-steel screw fixation in rats.

An increase in the mechanical fixation in bone of metallic biomaterials is considered advantageous in joint replacement and fracture surgery. Different approaches to improve fixation may be e.g. surface roughening, Ca-mineral coating or surface immobilization of growth factors or drugs. In the present work, bisphosphonate, a class of drugs that inhibit bone resorption, was immobilized onto stainless-steel screws. The screws were first roughened and coated with immobilized and cross-linked fibrinogen. Subsequently, an N-bisphosphonate, pamidronate, was immobilized onto fibrinogen, and another N-bisphosphonate, ibandronate, adsorbed on top of this. The so coated screws were inserted into the tibiae of eight male Sprague-Dawley rats. Another eight rats received screws prepared in the same way, but without the bisphosphonate coating. Pullout strength tests were performed after 2 weeks of implantation. The results showed a 28% (p=0.0009) higher pullout force and 90% increased pullout energy for the bisphosphonate coated screws, and support the idea that surface immobilized bisphosphonates can be used to improve biomaterials fixation in bone.

Peptide functionalized poly(L-lysine)-g-poly(ethylene glycol) on titanium: resistance to protein adsorption in full heparinized human blood plasma.

The graft copolymer poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) and its RGD- and RDG-functionalized derivatives (PLL-g-PEG/PEG-peptide) were assembled from aqueous solutions on titanium (oxide) surfaces. The polymers were characterized by NMR in order to determine quantitatively the grafting ratio, g (Lys monomer units/PEG side chains), and the fraction of the PEG side chains carrying the terminal peptide group. The titanium surfaces modified with the polymeric monomolecular adlayers were exposed to full heparinized blood plasma. The adsorbed masses were measured by in situ ellipsometry. The different PLL-g-PEG-coated surfaces showed, within the detection limit of the ellipsometric technique, no statistically significant protein adsorption during exposure to plasma for 30 min at 22 degrees C or 37 degrees C, whereas clean, uncoated titanium surfaces adsorbed approximately 350 ng/cm2 of plasma proteins. The high degree of resistance of the PEGylated surface to non-specific adsorption makes peptide-modified PLL-g-PEG a useful candidate for the surface modification of biomedical devices such as implants that are capable of eliciting specific interactions with integrin-type cell receptors even in the presence of full blood plasma. The results refer to short-term blood plasma exposure that cannot be extrapolated a priori to long-term clinical performance.

Penetration and loading of human serum albumin in porous silicon layers with different pore sizes and thicknesses.

Human serum albumin was adsorbed into porous silicon layers with thickness up to 3 microm and with different mean pore radius in the range 4.5-10 nm. The adsorbed amount of protein was quantified by I(125) radioactive labeling techniques and ellipsometry. The results show that albumin penetrated into the pores when the mean pore radius was larger than 5.5 nm, but could not totally occupy the available surface area when the layer thickness was larger than 1 microm. Loading of albumin both into porous layers and onto plane silicon as a function of albumin concentration was also investigated. These measurements show that loading of protein increased with protein concentration at least up to 10 mg/ml for porous silicon and up to 1 mg/ml for plane silicon. The maximum deposition into the type of porous layers used here was 28 microg/cm(2), compared to 0.36 microg/cm(2) for plane silicon.

Bone formation after 4 weeks around blood-plasma-modified titanium implants with varying surface topographies: an in vivo study.

The aim of the present study was to investigate and compare the stability and bone ingrowth capacity to screw-shaped titanium implants with five different surface treatments. The implants were: (1) standard turned with a thin blood plasma coat (TP), (2) NaOH-etched dito with pore size 0.2-0.3 microm (E), (3) NaOH-etched with pore size 0.2-0.3 microm and a thin blood plasma coat (EP), (4) electrochemically oxidised with pore size 1-2 microm (O), (5) electrochemically oxidised with pore size 1-2 microm and a thin blood plasma coat (OP). A total of 66 implants were divided into the above-described five groups and inserted for 4 weeks into tibia and femur of 11 rabbits. The implants were evaluated by resonance frequency (RF) measurements at the time of insertion and removal, and analysed histomorphometrically at removal. The RF measurements showed that the implant stability was lower in soft bone compared to dense and increased with time. No significant differences were observed between the different surface modifications. The histomorphometric analysis revealed no statistically significant differences between the implants regarding bone-to-metal contact (BMC) and bone area inside the threads (BA). The above results indicate that thin blood plasma-coated and non-coated screw-shaped titanium implants with turned, NaOH-etched and electrochemically etched surface profiles integrate similarly to bone at 1 month of implantation.

IL-1alpha, IL-1beta and TNF-alpha secretion during in vivo/ex vivo cellular interactions with titanium and copper.

Titanium (Ti) and copper (Cu) were used to evaluate cytokine secretion around materials with different chemical properties. Ti disks were coated with Cu or left uncoated. The disks were inserted subcutaneously in rats for 1, 3, 12, 18, 24 and 48 h. Interleukin-1alpha (IL-1alpha), IL-1beta and tumor necrosis factor-alpha (TNF-alpha) concentrations were measured in vivo around the materials, in sham operated sites, and after ex vivo incubation of surface adherent cells. Ti and Cu revealed distinct cytokine expression patterns. Cu recruited cells showed higher and prolonged release of IL-1alpha than Ti at longer times (>24 h), whereas Ti exhibited a transient IL-1alpha response at earlier periods (<24 h). An early enhanced secretion of TNF-alpha characterized Ti. Low amounts of IL-1beta were found around both materials. Sham site recruited cells produced lower levels of cytokines. The results after ex vivo incubations were similar to those in vivo. This study shows that material chemical properties influence early cytokine production. The Ti-associated transient rise of IL-1alpha and TNF-alpha may be of importance for the early tissue response around biocompatible materials, while a delayed high IL-1alpha expression could be a marker of inflammation induced by toxic materials.

C1q-independent activation of neutrophils by immunoglobulin M-coated surfaces.

Neutrophil granulocytes are known to rapidly adhere and undergo frustrated phagocytosis upon contact with immunoglobulin and/or complement protein opsonized artificial surfaces. In this study, we examined the relation between serum protein deposition and human neutrophil activation on hydrophobic glass and silicon model surfaces that were coated with immunoglobulin G or M (IgG/IgM), both initiators of the classical complement pathway. Protein adsorption from normal human serum (NHS) was quantified with null-ellipsometry combined with antibody techniques. The neutrophil oxygen radical production was registered by luminol-amplified chemiluminescence (CL) and the morphology, as well as changes in the content of filamentous actin (F-actin), were documented by fluorescence microscopy. Complement factor 3 (C3) bound to both IgG- and IgM-coated surfaces, but surprisingly C1q was found only on IgG-coated surfaces. Both immunoglobulins triggered complement dependent neutrophil activation. However, CL and F-actin accumulation were found sensitive to the presence of C1q in the serum only at the IgG-coated surface. We suggest that spontaneously adsorbed IgM activates the complement system and interacts with neutrophils by C1q-independent mechanisms.