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The aim of this study was to investigate the effect of TiO2 scaffold (SC) coated with an alginate hydrogel containing a proline-rich peptide (P2) on osteoblast proliferation and differentiation in vitro. Peptide release was evaluated and a burst release was observed during the first hours of incubation, and then progressively released overtime. No changes were observed in the cytotoxicity after 48 h of seeding MC3T3-E1 cells on the coated and uncoated TiO2 SC. The amount of cells after 7 days was higher on uncoated TiO2 SC than on alginate-coated TiO2 SC, measured by DNA content and scanning electron microscope imaging. In addition, while lower expression of integrin beta1 was detected for alginate-coated TiO2 SC at this time point, similar gene expression was observed for other integrins, fibronectin-1, and several osteoblast differentiation markers. After 21 days, gene expression of integrin beta3, fibronectin-1, osterix, and collagen-I was increased in alginate-coated compared to TiO2 SC. Moreover, increased gene expression of integrin alpha8, bone morphogenetic protein 2, interleukin-6, and collagen-I was found on P2 alginate-coated TiO2 SC compared to alginate-coated TiO2 SC. In conclusion, our results indicate that alginate-coated TiO2 SC can act as a matrix for delivery of proline-rich peptides increasing osteoblast differentiation. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013. [ABSTRACT FROM AUTHOR]

Background: Canine coxofemoral joint osteoarthritis is a common, painful and debilitating condition. The objective of this study was to evaluate if any measurable changes in pain or lameness occurred in this patient group immediately after a single treatment with pulsed electromagnetic field therapy. Eight dogs with coxofemoral joint osteoarthritis presenting with signs of pain and lameness were prospectively recruited to this randomised, controlled, double blinded, cross-over study. Subjects attended the research facility on two occasions for one active and one placebo treatment with pulsed electromagnetic field therapy. The immediate effect of one pulsed electromagnetic field therapy treatment on pain and lameness was measured subjectively with the Helsinki Chronic Pain Index and Visual Analogue Scale and objectively using a pressure sensitive walkway., Results: A statistically significant difference (P = 0.03) for change in stride length in the affected limb was recorded for subjects between the active and placebo treatments with pulsed electromagnetic field therapy. Within the active treatment results, there was a statistically significant change in the measurement for reach (P = 0.04) and stride length (P = 0.047) which got shorter in the affected limb post treatment. For the subjective outcome measures, there was no statistically significant difference between the active and placebo treatments for the evening of the treatment day or the next morning from pre-treatment values. Within the placebo treatment results a statistically significant change (improvement) was detected in Visual Analogue Score (P = 0.03) between pre-treatment and the next morning values., Conclusions: The findings of this study do not show demonstrable improvement in owner assessed pain levels or temporospatial performance in dogs with coxofemoral joint osteoarthritis immediately after a single application of pulsed electromagnetic field therapy., (© 2024. The Author(s).)

Nanocoatings based on plant polyphenols have been recently suggested as a potent strategy for modification of implant surfaces for enhancing host cell attachment and reducing bacterial colonisation. In this study we aimed to investigate how serum proteins impact the early adhesion dynamics of human gingival fibroblasts onto titanium surfaces coated with tannic acid (TA). Silicate-TA nanocoatings were formed on titanium and pre-conditioned in medium supplemented with 0, 0.1, 1 or 10% FBS for 1 hour. Dynamics of fibroblasts adhesion was studied using quartz crystal microbalance with dissipation (QCM-D). Time-lapse imaging was employed to assess cell area and motility, while immunofluorescence microscopy was used to examine cell morphology and focal adhesion formation. Our results showed that in serum-free medium, fibroblasts demonstrated enhanced and faster adhesion to TA coatings compared to uncoated titanium. Increasing the serum concentration reduced cell adhesion to nanocoatings, resulting in nearly complete inhibition at 10% FBS. This inhibition was not observed for uncoated titanium at 10% FBS, although cell adhesion was delayed and progressed slower compared to serum-free conditions. In addition, 1% FBS dramatically reduced cell adhesion on uncoated titanium. We revealed a positive relationship between changes in dissipation and changes in cell spreading area, and a negative relationship between dissipation and cell motility. In conclusion, our study demonstrated that serum decreases fibroblasts interaction with surfaces coated with TA in a concentration dependent manner. This suggests that controlling serum concentration can be used to regulate or potentially prevent fibroblasts adhesion onto TA-coated titanium surfaces.

Objectives: Access to the implant surface plays a significant role in effective mechanical biofilm removal in peri-implantitis treatment. Mechanical decontamination may also alter the surface topography of the implant, potentially increasing susceptibility to bacterial recolonization. This in vitro study aimed to evaluate a newly developed, anatomically realistic, and adaptable three-dimensional (3D)printed model with a peri-implant bone defect to evaluate the accessibility and changes of dental implant surfaces after mechanical decontamination treatment., Material and Methods: A split model of an advanced peri-implant bone defect was prepared using 3D printing. The function of the model was tested by mechanical decontamination of the exposed surface of dental implants (Standard Implant Straumann AG) coated with a thin layer of colored occlusion spray. Two different instruments for mechanical decontamination were used. Following decontamination, the implants were removed from the split model and photographed. Image analysis and fluorescence spectroscopy were used to quantify the remaining occlusion spray both in terms of area and total amount, while scanning electron microscopy and optical profilometry were used to analyze alteration in the implant surface morphology., Results: The 3D model allowed easy placement and removal of the dental implants without disturbing the implant surfaces. Qualitative and quantitative assessment of removal of the occlusion spray revealed differences in the mechanism of action and access to the implant surface between tested instruments. The model permitted surface topography analysis following the decontamination procedure., Conclusion: The developed 3D model allowed a realistic simulation of decontamination of implant surfaces with colored occlusion spray in an advanced peri-implant defect. 3D printing allows easy adaptation of the model in terms of the shape and location of the defect. The model presents a valuable tool for in vitro investigation of the accessibility and changes of the implant surface after mechanical and chemical decontamination., (© 2024 The Authors. Clinical and Experimental Dental Research published by John Wiley & Sons Ltd.)

Objective: The influence of radiation backscatter from titanium on DNA damage and migration capacity of human osteoblasts (OBs) and mesenchymal stem cells (MSCs) may be critical for the osseointegration of dental implants placed prior to radiotherapy. In order to evaluate effects of radiation backscatter, the immediate DNA damage and migration capacity of OBs and MSCs cultured on titanium or plastic were compared after exposure to ionizing irradiation., Materials and Methods: Human OBs and MSCs were seeded on machined titanium, moderately rough fluoride-modified titanium, or tissue culture polystyrene, and irradiated with nominal doses of 2, 6, 10, or 14 Gy. Comet assay was performed immediately after irradiation, while a scratch wound healing assay was initiated 24 h post-irradiation. Fluorescent live cell imaging documented the migration., Results: DNA damage increased with higher dose and with backscatter from titanium, and MSCs were significantly more affected than OBs. All doses of radiation accelerated the cell migration on plastic, while only the highest dose of 10 Gy inhibited the migration of both cell types on titanium., Conclusions: High doses (10 Gy) of radiation inhibited the migration capacity of both cell types on titanium, whereas lower doses (2 and 6 Gy) did not affect the migration of either OBs or MSCs., Clinical Relevance: Fractionated doses of 2 Gy/day, as distributed in conventional radiotherapy, appear not to cause severe DNA damage or disturb the migration of OBs or MSCs during osseointegration of dental implants., (© 2023. The Author(s).)

In vitro models of primary human osteocytes embedded in natural mineralized matrix without artificial scaffolds are lacking. We have established cell culture conditions that favored the natural 3D orientation of the bone cells and stimulated the cascade of signaling needed for primary human osteoblasts to differentiate into osteocytes with the characteristically phenotypical dendritic network between cells. Primary human osteoblasts cultured in a 3D rotating bioreactor and incubated with a combination of vitamins A, C, and D for up to 21 days produced osteospheres resembling native bone. Osteocyte-like cells were identified as entrapped, stellate-shaped cells interconnected through canaliculi embedded in a structured, mineralized, collagen matrix. These cells expressed late osteoblast and osteocyte markers such as osteocalcin (OCN), podoplanin (E11), dentin matrix acidic phosphoprotein 1 (DMP1), and sclerostin (SOST). Organized collagen fibrils, observed associated with the cell hydroxyapatite (HAp) crystals, were found throughout the spheroid and in between the collagen fibrils. In addition to osteocyte-like cells, the spheroids consisted of osteoblasts at various differentiation stages surrounded by a rim of cells resembling lining cells. This resemblance to native bone indicates a model system with potential for studying osteocyte-like cell differentiation, cross-talk between bone cells, and the mineralization process in a bonelike structure in vitro without artificial scaffolds. In addition, natural extracellular matrix may allow for the study of tissue-specific biochemical, biophysical, and mechanical properties. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research., (© 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.)

In head and neck cancer patients receiving dental implants prior to radiotherapy, backscatter from titanium increases the radiation dose close to the surface, and may affect the osseointegration. The dose-dependent effects of ionizing radiation on human osteoblasts (hOBs) were investigated. The hOBs were seeded on machined titanium, moderately rough fluoride-modified titanium, and tissue culture polystyrene, and cultured in growth- or osteoblastic differentiation medium (DM). The hOBs were exposed to ionizing γ-irradiation in single doses of 2, 6 or 10 Gy. Twenty-one days post-irradiation, cell nuclei and collagen production were quantified. Cytotoxicity and indicators of differentiation were measured and compared to unirradiated controls. Radiation with backscatter from titanium significantly reduced the number of hOBs but increased the alkaline phosphatase activity in both types of medium when adjusted to the relative cell number on day 21. Irradiated hOBs on the TiF-surface produced similar amounts of collagen as unirradiated controls when cultured in DM. The majority of osteogenic biomarkers significantly increased on day 21 when the hOBs had been exposed to 10 Gy, while the opposite or no effect was observed after lower doses. High doses reinforced with backscatter from titanium resulted in smaller but seemingly more differentiated subpopulations of osteoblasts., Competing Interests: One of the authors, J. E. Ellingsen, has a patent licensing agreement with Dentsply Sirona Implants. The other authors declare no conflict of interest., (© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Highly specialized enamel matrix proteins (EMPs) are predominantly expressed in odontogenic tissues and diverged from common ancestral gene. They are crucial for the maturation of enamel and its extreme complexity in multiple independent lineages. However, divergence of EMPs occured already before the true enamel evolved and their conservancy in toothless species suggests that non-canonical functions are still under natural selection. To elucidate this hypothesis, we carried out an unbiased, comprehensive phenotyping and employed data from the International Mouse Phenotyping Consortium to show functional pleiotropy of amelogenin, ameloblastin, amelotin, and enamelin, genes, i.e. in sensory function, skeletal morphology, cardiovascular function, metabolism, immune system screen, behavior, reproduction, and respiratory function. Mice in all KO mutant lines, i.e. amelogenin KO, ameloblastin KO, amelotin KO, and enamelin KO, as well as mice from the lineage with monomeric form of ameloblastin were affected in multiple physiological systems. Evolutionary conserved motifs and functional pleiotropy support the hypothesis of role of EMPs as general physiological regulators. These findings illustrate how their non-canonical function can still effect the fitness of modern species by an example of influence of amelogenin and ameloblastin on the bone physiology., (© 2023. The Author(s).)

Dental implants, usually made of titanium, are exposed to hostile oral microflora that facilitate bacterial infections and subsequent inflammation. To mitigate these processes, we coated titanium substrates with block copolymer nanopatterns and investigated the bactericidal effect of these coatings against Gram-positive and Gram-negative bacteria. We found that the bactericidal efficacy of the coatings depends on their morphology and surface chemistry as well as on the bacterial strain: an optimal combination can lead to significant bacterial death for a short time, i.e. 90% for 90 min. Human gingival fibroblasts in contact with the nanopatterned coatings showed similar cell attachment and morphology as on bare Ti. Immunostaining assays showed similar levels of CCR7 and CD206 in macrophages cultured over the nanopatterns and bare Ti, demonstrating adequate properties for tissue integration. The nanopatterns induced a small increase in macrophage aspect ratio, which might indicate early states of M2 polarization, given the absence of CD206.

Oral biofilms can be a major health problem causing infections and chronic inflammation of mucosal tissue. While much effort is put in the investigation of bacteria in biofilms, the role of fungi is often neglected, despite Candida albicans playing a key role in the formation of multispecies oral biofilms. With the rise of antibiotic resistance, new strategies to reduce microbial growth need to be found. Therefore, plant derived polyphenolic molecules have been suggested to reduce both adhesion and growth of pathogenic bacteria and fungi. In this study, we investigated the use of polyphenolic coatings to reduce adhesion and biofilm formation of C. albicans BWP17 on titanium implants. Tannic acid and pyrogallol coatings altered the hydrophobic and charge properties of titanium surfaces, and both compounds were gradually released as active molecules over time. Despite such effects, we found no significant inhibition on growth and biofilm formation of C. Albicans, indicating that the release of active molecules from the coatings did not reach relevant inhibitory concentrations. However, a potential antibiofilm effect was observed by the pH-dependent disassembly of the polyphenolic layer, which caused the biofilm to detach. Hence, further efforts are required to create tailored implant surfaces, which sustainably reduce microbial growth and adhesion., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The foreign body response is dictating the outcome of wound healing around any implanted materials. Patients who suffer from chronic inflammatory diseases and impaired wound healing often face a higher risk for implant failure. Therefore, functional surfaces need to be developed to improve tissue integration. For this purpose, we evaluated the impact of surface coatings made of antioxidant polyphenolic molecules tannic acid (TA) and pyrogallol (PG) on the host response in human blood. Our results showed that although the polyphenolic surface modifications impact the initial blood protein adsorption compared to Ti, the complement and coagulation systems are triggered. Despite complement activation, monocytes and granulocytes remained inactivated, which was manifested in a low pro-inflammatory cytokine expression. Under oxidative stress, both coatings were able to reduce intracellular reactive oxygen species in human gingival fibroblasts (hGFs). However, no anti-inflammatory effects of polyphenolic coatings could be verified in hGFs stimulated with lipopolysaccharide and IL-1β. Although polyphenols reportedly inhibit the NF-κB signaling pathway, phosphorylation of NF-κB p65 was observed. In conclusion, our results indicated that TA and PG coatings improved the hemocompatibility of titanium surfaces and have the potential to reduce oxidative stress during wound healing., (© 2022 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Background: Insufficient bone volume around an implant is a common obstacle when dental implant treatment is considered. Limited vertical or horizontal bone dimensions may lead to exposed implant threads following placement or a gap between the bone and implant. This is often addressed by bone augmentation procedures prior to or at the time of implant placement. This study evaluated bone healing when a synthetic TiO 2 block scaffold was placed in circumferential peri-implant defects with buccal fenestrations., Methods: The mandibular premolars were extracted and the alveolar bone left to heal for 4 weeks prior to implant placement in six minipigs. Two cylindrical defects were created in each hemi-mandible and were subsequent to implant placement allocated to treatment with either TiO 2 scaffold or sham in a split mouth design. After 12 weeks of healing time, the samples were harvested. Microcomputed tomography (MicroCT) was used to investigate defect fill and integrity of the block scaffold. Distances from implant to bone in vertical and horizontal directions, percentage of bone to implant contact and defect fill were analysed by histology., Results: MicroCT analysis demonstrated no differences between the groups for defect fill. Three of twelve scaffolds were partly fractured. At the buccal sites, histomorphometric analysis demonstrated higher bone fraction, higher percentage bone to implant contact and shorter distance from implant top to bone 0.5 mm lateral to implant surface in sham group as compared to the TiO 2 group., Conclusions: This study demonstrated less bone formation with the use of TiO 2 scaffold block in combination with implant placement in cylindrical defects with buccal bone fenestrations, as compared to sham sites., (© 2021. The Author(s).)

Platelet lysates (PL) contain a selection of proteins and growth factors (GFs) that are known to mediate cell activity. Many of these biomolecules have been identified as chemoattractants with the capacity to induce cell migration. In order to effectively deliver and retain these biomolecules to the site of injury, a scaffold containing PL could be an option. We use poly(ethylene glycol) (PEG) hydrogels consisting of 90 vol % PL to investigate their migratory potential on human mesenchymal stem cells (hMSCs). Cells exposed to these hydrogels were tracked, resulting in cell trajectories and detailed migratory parameters (velocity, Euclidean distance, directness, and forward migration index). Volumetric swelling ratios, hydrogel mechanical properties, and the release kinetics of proteins and GFs from hydrogels were also assessed. Furthermore, hMSC spheroids were encapsulated within the hydrogels to qualitatively assess cell invasion by means of sprouting and disintegration of the spheroid. Cell spheroids encapsulated within the PL-PEG gels exhibited initial outgrowths and eventually colonized the 3D matrix successfully. Results from this study confirmed that hMSCs exhibit directional migration toward the PL-loaded hydrogel with increased velocity and directness, compared to the controls. Overall, the incorporation of PL renders the PEG hydrogel bioactive. This study demonstrates the capacity of PL-loaded hydrogel constructs to attract stem cells for endogenous tissue engineering purposes.

Metallic implants are widely used in diverse clinical applications to aid in recovery from lesions or to replace native hard tissues. However, the lack of integration of metallic surfaces with soft tissue interfaces causes the occurrence of biomaterial-associated infections, which can trigger a complicated inflammatory response and, ultimately, implant failure. Here, a multifunctional implant surface showing nanoscale anisotropy, based on the controlled deposition of cellulose nanocrystals (CNC), and biological activity derived from platelet lysate (PL) biomolecules sequestered and presented on CNC surface, is proposed. The anisotropic radial nanopatterns are produced on polished titanium surfaces by spin-coating CNC at high speed. Furthermore, CNC surface chemistry allows to further sequester and form a coating of bioactive molecules derived from PL. The surface anisotropy provided by CNC guides fibroblasts growth and alignment up to 14 days of culture. Moreover, PL-derived biomolecules polarize macrophages toward the M2-like anti-inflammatory phenotype. These results suggest that the developed multifunctional surfaces can promote soft tissue integration to metallic implants and, at the same time, prevent bacterial invasion, tissue inflammation, and failure of biomedical metallic implants., (© 2021 Wiley-VCH GmbH.)

Objective: To compare surface topography of porcine and human root dentin and to develop a new in vitro model for class II furcation defects. The hypothesis for this study was that porcine mandible blocks can function as a model for class II furcation defects., Background: Treatment of mandibular class II furcation defects is unpredictable. There is a need for in vitro models to investigate new treatment methods., Methods: A model to investigate the surface topography of porcine and human root dentin was developed and the two tissues compared by SEM imaging and profilometer. A novel method for studying class II furcation defects was then tested. Blocks of porcine mandibles with molar 3 were prepared. Buccal class II furcation defects were created. The furcation area was isolated and bioluminescent Staphylococcus epidermidis Xen43 was used to form a biofilm in the furcation area to test the functionality of the novel furcation model., Results: Micromechanical damage caused by debridement on porcine and human root dentin showed similar pattern. No significant difference in the surface morphological parameters was observed between the corresponding porcine and human samples. The model allowed for assessment of the root surface inside the furcation area. While the number of viable bacteria in the furcation following debridement could be quantified, no significant difference between the treatment groups was detected, likely due to bacterial colonization within the periodontal ligament space., Conclusion: Porcine and human root dentin show similar surface topography following surface debridement. Porcine mandible blocks can function as a model for class II furcation defects. However, further development and refinement of the novel in vitro model is warranted., (© 2020 The Authors. Clinical and Experimental Dental Research published by John Wiley & Sons Ltd.)

Polyphenolic molecules have become attractive building blocks for bioinspired materials due to their adhesive characteristics, capacity to complex ions, redox chemistry, and biocompatibility. For the formation of tannic acid (TA) surface modifications based on silicate-phenolic networks, a high ionic strength is required. In this study, we investigated the effects of NaCl, KCl, and LiCl on the formation of TA coatings and compared it to the coating formation of pyrogallol (PG) using a quartz-crystal microbalance. We found that the substitution of NaCl with KCl inhibited the TA coating formation through the high affinity of K + to phenolic groups resulting in complexation of TA. Assessment of the radical formation of TA by electron paramagnetic resonance spectroscopy showed that LiCl resulted in hydrolysis of TA forming gallic acid radicals. Further, we found evidence for interactions of LiCl with the Si aq crosslinker. In contrast, the coating formation of PG was only little affected by the substitution of NaCl with LiCl or KCl. Our results demonstrate the interaction potential between alkali metal salts and phenolic compounds and highlight their importance in the continuous deposition of silicate-phenolic networks. These findings can be taken as guidance for future biomedical applications of silicate-phenolic networks involving monovalent ions.

Synthetic hydrogel-amorphous calcium phosphate composites are promising candidates to substitute biologically sourced scaffolds for bone repair. While the hydrogel matrix serves as a template for stem cell colonisation, amorphous calcium phosphate s provide mechanical integrity with the potential to stimulate osteogenic differentiation. Here, we utilise composites of poly(ethylene glycol)-based hydrogels and differently stabilised amorphous calcium phosphate to investigate potential effects on attachment and osteogenic differentiation of human mesenchymal stem cells. We found that functionalisation with integrin binding motifs in the form of RGD tripeptide was necessary to allow adhesion of large numbers of cells in spread morphology. Slow dissolution of amorphous calcium phosphate mineral in the scaffolds over at least 21 days was observed, resulting in the release of calcium and zinc ions into the cell culture medium. While we qualitatively observed an increasingly mineralised extracellular matrix along with calcium deposition in the presence of amorphous calcium phosphate-loaded scaffolds, we did not observe significant changes in the expression of selected osteogenic markers., Competing Interests: Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2020.)

Bone tissue regeneration in critical-size defects is possible after implantation of a 3D scaffold and can be additionally enhanced once the scaffold is enriched with drugs or other factors supporting bone remodelling and healing. Sodium alendronate (Aln), a widely used anti-osteoporosis drug, exhibits strong inhibitory effect on bone resorption performed by osteoclasts. Thus, we propose a new approach for the treatment of bone defects in craniofacial region combining biocompatible titanium dioxide scaffolds and poly(l-lactide- co -glycolide) microparticles (MPs) loaded with Aln. The MPs were effectively attached to the surface of the scaffolds' pore walls by human recombinant collagen. Drug release from the scaffolds was characterized by initial burst (24 ± 6% of the drug released within first 24 h) followed by a sustained release phase (on average 5 µg of Aln released per day from Day 3 to Day 18). In vitro tests evidenced that Aln at concentrations of 5 and 2.5 µg/ml was not cytotoxic for MG-63 osteoblast-like cells (viability between 81 ± 6% and 98 ± 3% of control), but it prevented RANKL-induced formation of osteoclast-like cells from macrophages derived from peripheral blood mononuclear cells, as shown by reduced fusion capability and decreased tartrate-resistant acid phosphatase 5b activity (56 ± 5% reduction in comparison to control after 8 days of culture). Results show that it is feasible to design the scaffolds providing required doses of Aln inhibiting osteoclastogenesis, reducing osteoclast activity, but not affecting osteoblast functions, which may be beneficial in the treatment of critical-size bone tissue defects., (© The Author(s) 2020. Published by Oxford University Press.)

Surface modification with polyphenolic molecules has been pursued in biomedical materials owing to their antioxidant, anti-inflammatory, and antimicrobial characteristics. Recently, the use of silicic acid (Si aq ) as a mediator for efficient surface deposition of tannic acid (TA) was reported, but the postulated Si-TA polymeric networks were not characterized. Herein, we present unambiguous evidence for silicate-TA networks that involve Si-O-C motifs by using solid-state NMR spectroscopy, further supported by XPS and ToF-SIMS. By using QCM-D we demonstrate the advantages of Si aq , compared to using transition-metal ions, to improve the coating efficiency under mildly acidic conditions. The presented homogenous coating buildup and validated applicability in inorganic buffers broadens the use of TA for surface modifications in technological and biomedical applications., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

In the quest for finding new strategies to enhance tissue integration and to reduce the risk of bacterial colonization around endosseous implants, we report the application of auto-oxidative phenolic coatings made of tannic acid and pyrogallol to titanium surfaces. The functionalized surfaces were screened for their biological performance using cultures of primary human osteoblasts and biofilm-forming bioluminescent staphylococci S. epidermidis Xen43 and S. aureus Xen29. No toxic effect of the coatings on osteoblasts was detected. While tannic acid coatings seemed to induce a delay in osteoblast maturation, they revealed anti-inflammatory potential. Similar effects were observed for pyrogallol coatings deposited for 24 h. Thin pyrogallol coatings deposited for 2 h seemed to promote osteoblast maturation and revealed increased calcium deposition. The effects on osteoblast were found to be related to the release of phenolic compounds from the surfaces. While the phenolic coatings could not inhibit staphylococcal biofilm formation on the titanium surfaces, released phenolic compounds had an inhibitory effect the growth of planktonic bacteria. In conclusion, the assessed coating systems represent a versatile functionalization method which exhibit promising effects for endosseous implant applications.

Acellular polymer-calcium phosphate composites are promising bone graft materials. Hydrogels are suitable for providing a temporary matrix, while calcium phosphate minerals serve as ion depots for calcium and phosphate required for de novo bone formation. Crystalline calcium phosphates are stable under biological conditions and are commonly used in such scaffolds. However, the low solubility of these phases reduces the availability of free ions and potentially obstructs the remodelling necessary for the formation of mineralised tissue. Here, we investigate two different strategies to stabilise amorphous calcium phosphates in a synthetic polyethylene glycol-based hydrogel matrix. In vitro experiments mimicking an injectable application showed that amorphous calcium phosphate (ACP) of variable stability was formed in the hydrogel matrices. In additive-free composites, ACP transformed into brushite within minutes. Citrate or zinc additives were found to stabilise the formed ACP phase to different degrees. In the presence of citrate, ACP was stable for at least 2 h before it transformed into hydroxyapatite within 3-20 days. Partial calcium substitution with zinc (Zn/Ca = 10%) produced zinc-doped ACP of high stability that did not show signs of crystallisation for at least 20 days. The presented methods and findings open new possibilities for the design of novel injectable synthetic bone graft materials. The possibility to produce ACP with tailorable stability promises great potential for creating temporary scaffolds with good osteogenic properties. STATEMENT OF SIGNIFICANCE: Synthetic hydrogel-calcium phosphate (CaP) composites are promising biomaterials to replace human- and animal-derived bone scaffolds. Most reported hydrogel-CaP composite materials employ crystalline CaP phases that lack the osteoinductive properties of autograft. Stabilising amorphous calcium phosphates (ACP) could overcome this limitation, readily delivering calcium and phosphate ions and facilitating remodelling into new bone tissue. The design of synthetic hydrogel-ACP scaffolds, however, requires more understanding of the mineralisation processes in such matrices. This study presents a model system to characterise the complex mineral formation and transformation processes within a hydrogel matrix. We demonstrate a facile route to produce self-mineralising injectable synthetic hydrogels and prove two different strategies to stabilise ACP for different periods within the formed composites., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Tannic acid (TA) adheres to a broad variety of different materials and forms versatile surface coatings for technical and biological applications. In mild alkaline conditions, autoxidation processes occur and a firm monolayer is formed. Up to now, thicker coatings are obtained in only a cross-linked multilayer fashion. This study presents an alternative method to form continuous TA coatings using orthosilicic acid (Si aq ). Adsorption kinetics and physical properties of TA coatings in the presence of Si aq were determined using a quartz-crystal microbalance and nanoplasmonic spectroscopy. An in situ TA layer thickness of 200 nm was obtained after 24 h in solutions supplemented with 80 μM Si aq . Dry-state measurements indicated a highly hydrated layer in situ. Furthermore, chemical analysis by Fourier transform infrared spectroscopy revealed possible complexation of TA by Si aq , whereas UV-vis spectroscopy did not indicate an interaction of Si aq in the autoxidation process of TA. Investigation of additional metalloid ions showed that germanic acid was also able to initiate a continuous coating formation of TA, whereas boric acid prevented the polymerization process. In comparison to that of TA, the coating formation of pyrogallol (PG) and gallic acid (GA) was not affected by Si aq . PG formed continuous coatings also without Si aq , whereas GA formed only a monolayer in the presence of Si aq . However, Si aq induced a continuous layer formation of ellagic acid. These results indicate the specific importance of orthosilicic acid in the coating formation of polyphenolic molecules with multiple ortho-dihydroxy groups and open new possibilities to deposit TA on interfaces.

TiO 2 scaffolds have previously shown to have promising osteoconductive properties in previous in vivo experiments. Appropriate mechanical stimuli can further promote this osteoconductive behaviour. However, the complex mechanical environment and the mechanical stimuli enhancing bone regeneration for porous bioceramics have not yet been fully elucidated. This paper aims to compare and evaluate mechanical environment of TiO 2 scaffold with three commercial CaP biomaterials, i.e. Bio-Oss, Cerabone and Maxresorb under simulated perfusion culture conditions. The solid phase and fluid phase were modelled as linear elastic material and Newtonian fluid, respectively. The mechanical stimulus was analysed within these porous scaffolds quantitatively. The results showed that the TiO 2 had nearly heterogeneous stress distributions, however lower effective Young's modulus than Cerabone and Maxresorb. The permeability and wall shear stress (WSS) for the TiO 2 scaffold was significantly higher than other commercial bone substitute materials. Maxresorb and Bio-Oss showed lowest permeability and local areas of very high WSS. The detailed description of the mechanical performance of these scaffolds could help researchers to predict cell behaviour and to select the most appropriate scaffold for different in vitro and in vivo performances. Graphical abstract Schematic representation of the establishment procedure. Take the establishment process of Cerabone as an example. Left shows a slice of micro-CT image from Cerabone, and 1.5 mm × 1.5 mm region of interest was shown in the red box. A 1.5-mm 3 cube was cut out by Boolean operation in Mimics (Materialise, Belgium), and the cubic model was remeshed in 3-Matic 6.0 (Materialise, Belgium). The cubic model is shown in blue, and the empty space in red.

Biomaterials which promote tissue integration and resist microbial colonisation are required in bone tissue engineering to prevent biomaterial-associated infections. Surface modification of established materials for bone tissue engineering, such as TiO 2 , have emerged as promising anti-infective strategies. Interestingly, the antibacterial activity of TiO 2 in the form of particles can be enhanced by combining it with H 2 O 2 , even in the absence of irradiation. However, it remains unknown whether TiO 2 surfaces elicit a similar effect. In this study, the antibacterial effect of porous TiO 2 scaffolds generated by the catalytic decomposition of H 2 O 2 in the absence of light (dark catalysis) was investigated. Porous ceramic foams were fabricated and sol-gel coated for high catalytic activity. Degradation of methylene blue in the presence of 3% H 2 O 2 increased by 80% for the sol-gel-coated surfaces. The degradation kinetics indicate that intermediate free radicals that form at the liquid-TiO 2 interface are responsible for the oxidative behavior of the surface. TiO 2 surfaces were further pretreated with 30% H 2 O 2 for prolonged oxidative behavior. The biological response toward such surfaces was assessed in vitro. S. epidermidis biofilms formed on modified surfaces showed reduced viability compared to nonmodified surfaces. Further, the same surface modification showed no cytotoxic effects on MC3T3 preosteoblasts. However, the results from the conducted genotoxicity assay were inconclusive, and further studies are needed to exclude ROS-mediated DNA damage. To conclude, this study provides evidence that a simple surface modification based on the dark catalytic effect of TiO 2 can be used to create antibacterial surface properties for ceramic bone scaffolds.

Hybrid poly(ethylene glycol)-co-peptide hydrogels are a versatile platform for bone regeneration. For the use as injectable scaffolds, a good understanding of reaction kinetics and physical properties is vital. However, these factors have not yet been comprehensively illuminated. We show that gelation time can be effectively controlled by pH without affecting the elasticity of the formed hydrogels. Maleimide functionalised PEG gels at lower pH and produces more densely cross-linked networks than vinylsulfone functionalised PEG. Both form non-ideal networks. The elastic moduli on the order of a few kPa are in good agreement with the structural characterisation. Primary human osteoblasts cultured in proximity to bulk gels were not adversely affected in vitro. The results demonstrate that hybrid PEG-peptide hydrogels can be tailored to the requirements of in situ gelation. Attributed to their increased structural properties and a higher tolerance towards low pH, maleimide functionalised hydrogels might provide a better alternative for injectable applications., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Strategies that enable hydrogel substrates to support cell attachment typically incorporate either entire extracellular matrix proteins or synthetic peptide fragments such as the RGD (arginine-glycine-aspartic acid) motif. Previous studies have carefully analysed how material characteristics can affect single cell morphologies. However, the influence of substrate stiffness and ligand presentation on the spatial organisation of human mesenchymal stem cells (hMSCs) have not yet been examined. In this study, we assessed how hMSCs organise themselves on soft (E = 7.4-11.2 kPa) and stiff (E = 27.3-36.8 kPa) poly(ethylene glycol) (PEG) hydrogels with varying concentrations of RGD (0.05-2.5 mM). Our results indicate that hMSCs seeded on soft hydrogels clustered with reduced cell attachment and spreading area, irrespective of RGD concentration and isoform. On stiff hydrogels, in contrast, cells spread with high spatial coverage for RGD concentrations of 0.5 mM or higher. In conclusion, we identified that an interplay of hydrogel stiffness and the availability of cell attachment motifs are important factors in regulating hMSC organisation on PEG hydrogels. Understanding how cells initially interact and colonise the surface of this material is a fundamental prerequisite for the design of controlled platforms for tissue engineering and mechanobiology studies., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Primary human osteoblasts and osteoclasts incubated in a rotating coculture system without any scaffolding material, form bone-like tissue that may be used to evaluate effects of various compounds on mechanical strength. Circulating adiponectin has been found to be negatively associated with BMD and strength and was therefore assessed in this system. Osteospheres of human osteoblasts and osteoclasts were generated with and without adiponectin. The osteospheres were scanned using micro-computed tomography, the mechanical properties were tested by flat punch compression using nanoindentation equipment, and the cellular morphology characterized by microscopy. The association between autologously produced adiponectin and biomechanical properties was further evaluated by quantitation of adiponectin levels using quantitative polymerase chain reaction (qPCR) and immunoassays, and identification of stiffness by bending test of rat femurs. The molecular mechanisms were examined in vitro using human bone cells. Mechanical testing revealed that adiponectin induced a more compliant osteosphere compared with control. The osteospheres had a round, lobulated appearance with morphologically different areas; inner regions containing few cells embedded in a bone-like material surrounded by an external area with a higher cell quantity. The expression of adiponectin was found to correlate positively to ultimate bending moment and ultimate energy absorption and deflection, on the other hand, it correlated negatively to bending stiffness, indicating autocrine and/or paracrine effects of adiponectin in bone. Adiponectin enhanced proliferation and expression of collagen, leptin, and tumor necrosis factor-alpha in osteoblasts and stimulated proliferation, but not the functional activity of osteoclasts. Our results indicate that both administration of adiponectin during osteosphere production and in situ elevated levels of adiponectin in rat femurs, reduced stiffness of the bone tissues. An increase in undifferentiated cells and extracellular matrix proteins, such as collagen, may explain the reduced bone stiffness seen in the osteospheres treated with adiponectin.

The chemical decontamination of infected dental implants is essential for the successful treatment of peri-implantitis. The aim of this study was to assess the antibacterial effect of a hydrogen peroxide-titanium dioxide (H 2 O 2 -TiO 2 ) suspension against Staphylococcus epidermidis biofilms. Titanium (Ti) coins were inoculated with a bioluminescent S. epidermidis strain for 8 h and subsequently exposed to H 2 O 2 with and without TiO 2 nanoparticles or chlorhexidine (CHX). Bacterial regrowth, bacterial load and viability after decontamination were analyzed by continuous luminescence monitoring, live/dead staining and scanning electron microscopy. Bacterial regrowth was delayed on surfaces treated with H 2 O 2 -TiO 2 compared to H 2 O 2 . H 2 O 2 -based treatments resulted in a lower bacterial load compared to CHX. Few viable bacteria were found on surfaces treated with H 2 O 2 and H 2 O 2 -TiO 2 , which contrasted with a uniform layer of dead bacteria for surfaces treated with CHX. H 2 O 2 -TiO 2 suspensions could therefore be considered an alternative approach in the decontamination of dental implants.

Bone scaffolds are susceptible for bacterial infection when implanted, particularly in compromised bone. Therefore anti-bacterial bone scaffolds are desirable. Here a novel approach to provide bactericidal properties for titanium dioxide scaffolds is proposed. Gentamicin loaded poly(L-lactide-co-glycolide) microparticles were immobilized on the scaffold pore walls by sodium alginate hydrogel. The results show that the microparticles were effectively immobilized on the scaffolds. Desired burst release was observed within the first 8h and gentamicin dose reached 125μg from single scaffold that corresponded to ~25% of total drug introduced in the system. Following the initial burst, the dose was gradually decreasing up to day 10 and afterwards a sustained release of 3μg/day was measured. Cumulatively ~90% of the drug was delivered up to day 50. Above pattern, i.e. burst release with following sustained release, is desired for prevention of perioperative bone infections: burst release stops local infections during post-implantation "decisive period" while further sustained drug release prevents bacterial recolonization. In vitro studies confirmed antimicrobial activity of released gentamicin against Staphylococcus spp. and cytocompatibility of the system with osteoblast-like cells (MG-63). Thus the system is a viable option for the treatment of bone tissue defects., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objectives: The primary objective was to assess osseointegration of implants with dehiscence defects grafted with a TiO 2 scaffold. The secondary objective was to assess the performance of the scaffold in terms of mechanical stability and bone fill., Material and Methods: Five minipigs had the mandibular premolars extracted. After healing, two dental implants (SLActive ® , Institut Straumann AG, Basel, Switzerland) with associated semi-cylindrical dehiscence defects (Ø = 6 mm, height = 10 mm) were installed in each quadrant. The defects were grafted with test scaffolds (n = 10) or control autologous bone blocks (n = 10). After 3 months submerged healing, the pigs were euthanized and the sites analysed by microcomputed tomography and histology., Results: Four minipigs were available for second stage surgery; (n = 9) experimental and (n = 7) control sites. The mean bone-to-implant contact on the defect side was 82% (±10%) and 79% (±11%) in the test and control groups respectively. The mean level of first bone-to-implant contact was more coronal on the defect side in the test group 3.2 mm (±0.4 mm) than in the control group 3.6 mm (±1.1 mm). The defect area occupied by bone within the extent of the scaffold varied, but averaged 37% (±14.6%) whereas the material itself occupied 7.4% (±3.5%)., Conclusions: Within the limitations of the study, the results suggest that the novel synthetic scaffold material perform similar to the autologous bone block control with respect to implant osseointegration. The mechanical properties of the scaffold appeared sufficient to withstand clinical load in the present experimental model., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Polyphenols can form functional coatings on a variety of different materials through auto-oxidative surface polymerization in a manner similar to polydopamine coatings. However, the mechanisms behind the coating deposition are poorly understood. We report the coating deposition kinetics of the polyphenol tannic acid (TA) and the simple phenolic compound pyrogallol (PG) on titanium surfaces. The coating deposition was followed in real time over a period of 24 h using a quartz crystal microbalance with dissipation monitoring (QCM-D). TA coatings revealed a multiphasic layer formation: the deposition of an initial rigid layer was followed by the buildup of an increasingly dissipative layer, before mass adsorption stopped after approximately 5 h of coating time. The PG deposition was biphasic, starting with the adsorption of a nonrigid viscoelastic layer which was followed by layer stiffening upon further mass adsorption. Coating evaluation by ellipsometry and AFM confirmed the deposition kinetics determined by QCM-D and revealed maximum coating thicknesses of approximately 50 and 75 nm for TA and PG, respectively. Chemical characterization of the coatings and polymerized polyphenol particles indicated the involvement of both physical and chemical interactions in the auto-oxidation reactions.

X-ray microtomography (micro-CT), one of the most resourceful instruments for high resolution 3D analysis, can provide qualitative and quantitative accurate structural and compositional information for a broad range of materials. Yet its contribution to the field of biopolymeric materials science is often limited by low imaging contrast due to scarce X-ray attenuation features, particularly for sponges and foam-like structures. This limitation can be overcome to some extent by adjusting the working parameters of micro-CT equipment. However, such approach also facilitates noise and artefacts, and solving the signal-to-noise trade-off has been always problematic. Searching for alternatives turns one's attention towards the improvement of X-ray attenuation features. While several studies report the use of contrast agents for biological materials, studies to integrate multiple micro-CT approaches for biopolymers were not conducted so far. This method paper is thus aimed to serve as a platform for micro-CT analysis of low X-ray absorptive polymers. Here, several contrast enhancing artifices were developed and trialled on gelatin and poly(vinyl alcohol) biopolymer composites (GP). Accordingly, GP were modified with iodine, barium, silver-based chemicals and hexa(methyl disilazane) by two different methods, i.e. addition of high atomic number chemicals during materials synthesis and post-synthesis staining, respectively. Consequently, cross-sectional scanning electron microscopy emerged as complementary characterization, aimed to confirm the reproducibility of samples morphological features. The most versatile methods were barium chloride additive incorporation and iodine staining coupled with hexa(methyl disilazane) chemical drying. Both methods significantly improved the X-ray absorbance of our polymeric samples, providing better contrast of micro-CT tomograms.

Cathodic polarization has been reported to enhance the ability of titanium based implant materials to interact with biomolecules by forming titanium hydride at the outermost surface layer. Although this hydride layer has recently been suggested to allow the immobilization of the broad spectrum antibiotic doxycycline on titanium surfaces, the involvement of hydride in binding the biomolecule onto titanium remains poorly understood. To gain better understanding of the influence this immobilization process has on titanium surfaces, mirror-polished commercially pure titanium surfaces were cathodically polarized in the presence of doxycycline and the modified surfaces were thoroughly characterized using atomic force microscopy, electron microscopy, secondary ion mass spectrometry, and angle-resolved X-ray spectroscopy. We demonstrated that no hydride was created during the polarization process. Doxycycline was found to be attached to an oxide layer that was modified during the electrochemical process. A bacterial assay using bioluminescent Staphylococcus epidermidis Xen43 showed the ability of the coating to reduce bacterial colonization and planktonic bacterial growth., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Biofilm formation on dental abutment may lead to peri-implant mucositis and subsequent peri-implantitis. These cases are clinically treated with antibiotics such as doxycycline (Doxy). Here we used an electrochemical method of cathodic polarization to coat Doxy onto the outer surface of a dental abutment material. The Doxy-coated surface showed a burst release in phosphate-buffered saline during the first 24 h. However, a significant amount of Doxy remained on the surface for at least 2 weeks especially on a 5 mA-3 h sample with a higher Doxy amount, suggesting both an initial and a long-term bacteriostatic potential of the coated surface. Surface chemistry was analyzed by x-ray photoelectron spectroscopy and secondary ion mass spectrometry. Surface topography was evaluated by field emission scanning electron microscopy and blue-light profilometry. Longer polarization time from 1 h to 5 h and higher current density from 1 to 15 mA cm(-2) resulted in a higher amount of Doxy on the surface. The surface was covered by a layer of Doxy less than 100 nm without significant changes in surface topography. The antibacterial property of the Doxy-coated surface was analyzed by biofilm and planktonic growth assays using Staphylococcus epidermidis. Doxy-coated samples reduced both biofilm accumulation and planktonic growth in broth culture, and also inhibited bacterial growth on agar plates. The antibacterial effect was stronger for samples of 5 mA-3 h coated with a higher amount of Doxy compared to that of 1 mA-1 h. Accordingly, an abutment surface coated with Doxy has potential for preventing bacterial colonization when exposed to the oral cavity. Doxy-coating could be a viable way to control peri-implant mucositis and prevent its progression into peri-implantitis.

In order to prevent soft tissue down-growth into osseous defect areas, membranes are used when placing bone graft materials. These membranes still show shortcomings in their performance and applications. In the current study, we choose an approach to integrate micro-porous surface structures into a macro-porous scaffold. Low porous surfaces were fabricated by dip-coatings. Four different material compositions (titanium dioxide, polycaprolactone, polycaprolactone/water, polycaprolactone/β-tricalcium phosphate) were characterised in terms of their appearance, architecture, topographical features and cell response. Titanium dioxide surfaces exhibited rougher and more complex textures, resulting in the highest number of osteosarcoma cells and distinct morphologies in terms of cell spreading. Polycaprolactone-based surfaces showed a smoother topography and enhanced microporosity, but the effect on secretion of the bone markers sclerostin and interleukin-6 from human osteoblasts was lower compared to secretion from cells cultured on titanium dioxide. β-Tricalcium phosphate modification of polycaprolactone did not show any significant improvement regarding cell-material interaction. Nevertheless, surfaces show potential in the mechanical blockage of epithelial and soft tissue cells and may still permit sufficient nutrient transport., (© The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.)

The purpose of bone tissue engineering is to employ scaffolds, cells, and growth factors to facilitate healing of bone defects. The aim of this study was to assess the viability and osteogenic differentiation of primary human osteoblasts and adipose tissue-derived mesenchymal stem cells from various donors on titanium dioxide (TiO2) scaffolds coated with an alginate hydrogel enriched with enamel matrix derivative. Cells were harvested for quantitative reverse transcription polymerase chain reaction on days 14 and 21, and medium was collected on days 2, 14, and 21 for protein analyses. Neither coating with alginate hydrogel nor alginate hydrogel enriched with enamel matrix derivative induced a cytotoxic response. Enamel matrix derivative-enriched alginate hydrogel significantly increased the expression of osteoblast markers COL1A1, TNFRSF11B, and BGLAP and secretion of osteopontin in human osteoblasts, whereas osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells seemed unaffected by enamel matrix derivative. The alginate hydrogel coating procedure may have potential for local delivery of enamel matrix derivative and other stimulatory factors for use in bone tissue engineering.

The use of hydraulic calcium phosphate cements (CPCs) as bone substitute is impaired by their relatively poor handling due to the need to mix a powder and a liquid during surgery. The aim of the present study was to assess the possibility to design CPCs as inorganic dual-paste cements, where both pastes would be stable for years, but would react as soon as they are mixed together. Results showed that aqueous pastes of α-tricalcium phosphate (α-TCP) powder could be stabilized for up to a year at room temperature by the use of 0.1 M Mg chloride solution. Adding a calcium chloride solution in a 1:4 volume ratio activated α-TCP pastes provided the Ca/Mg ratio was larger than one. Mechanistic investigations suggest that Ca ions can displace Mg cations adsorbed at the surface of α-TCP particles to initiate α-TCP transformation to calcium-deficient hydroxyapatite and concomitant paste hardening. The compressive strength (29 MPa) was similar to that of commercial formulations (5-80 MPa). Other divalent cations (Ba, Ni, Sr) had a similar effect although with a different degree of efficacy.

In this study the attempts to improve mechanical properties of highly-porous titanium dioxide scaffolds produced by polymer sponge replication method were investigated. Particularly the effect of two-step sintering at different temperatures on microstructure and mechanical properties (compression test) of the scaffolds were analysed. To this end microcomputed tomography and scanning electron microscopy were used as analytical methods. Our experiments showed that the most appropriate conditions of manufacturing were when the scaffolds were heat-treated at 1500 °C for 1 h followed by sintering at 1200 °C for 20 h. Such scaffolds exhibited the highest compressive strength which was correlated with the highest linear density and the lowest size of grains. Moreover, grain size distribution was narrower with predominating fraction of fine grains 10-20 μm in size. Smaller grains and higher linear density sug- gested that in this case densification process prevailed over undesirable process of grain coarsening, which finally resulted in im- proved mechanical properties of the scaffolds.