Development of a Coculture Model for Assessing Competing Host Mammalian Cell and Bacterial Attachment on Zirconia versus Titanium.

Objectives: Coculture models are limited by bacteria rapidly outcompeting host mammalian cells for nutrients in vitro, resulting in mammalian cell death. The goal of this study was to develop a coculture model enabling survival of mammalian cells and oral bacterial species to assess their competition for growth on dental implant materials. Methods: Two early colonizing oral bacterial species, Streptococcus mutans or Actinomyces naeslundii, were grown in coculture with primary human macrophages or human gingival fibroblasts for up to 7 days on tissue-culture treated polystyrene or polished titanium and zirconia disks. Chloramphenicol was supplemented in cell culture medium at bacteriostatic concentrations to maintain stable bacterial inoculum size. Planktonic and adherent bacterial growth was assessed via spot plating while mammalian cell growth and attachment were evaluated using colorimetric metabolic assay and confocal fluorescence microscopy, respectively. Results: Macrophages and fibroblasts proliferated in the presence of S. mutans and maintained viability above 70% during coculture for up to 7 days on tissue-culture treated polystyrene and polished titanium and zirconia. In contrast, both mammalian cell types exhibited decreasing proliferation and surface coverage on titanium and zirconia over time in coculture with A. naeslundii versus control. S. mutans and A. naeslundii were maintained within an order of magnitude of seeding inoculum sizes throughout coculture. Significance: Cell culture medium supplemented with antibiotics at bacteriostatic concentrations can suppress bacterial overgrowth and facilitate mammalian cell viability in coculture model systems. Within the study's limitations, oral bacteria and mammalian cell growth in coculture are comparable on polished titanium and zirconia surfaces.

A microtomographic and histopathological evaluation of dental cements as late-stage peri-implant complication in a rat model.

Cement mediated peri-implantitis accounts for 1.9-75% of dental implant failures associated with peri-implant diseases. This study evaluated the biological impact of dental cements on osseointegrated implants using Lewis rats. Twenty-two rats were distributed into 6 groups: negative control (NC) soft diet (SD), and hard diet (HD); positive control SD and HD (n = 3); Implant + bio-ceramic Cement (BC) SD and HD which included contralateral Sham sites (n = 5). Titanium implants were placed on either side of the maxillae and allowed to heal for 14 days. Later, both sides of experimental groups underwent a re-entry surgery to simulate clinical cementation. The right side received 0.60 mg of BC. At 14 days post cement application, maxillae were harvested for clinical, microtomographic, and histological evaluations. Clinical and microtomographic evaluations indicated evidence of extensive inflammation and circumferential bone resorption around BC implants in comparison to NC. Histology revealed cement particles surrounded by inflammatory infiltrate in the implant area accompanied by biofilm for SD groups. Both sides of BC indicated intensive bone resorption accompanied by signs of osteolysis when compared to NC. Cemented groups depicted significantly lower bone to implant contact when compared to NC. In conclusion, residual cement extravasation negatively impacted osseointegrated implants after re-entry surgeries.

A Model of Immediate Implant Placement to Evaluate Early Osseointegration in 129/Sv Diabetic Mice.

PURPOSE: To analyze the process of early oral osseointegration of titanium (Ti) implants in diabetic 129/Sv mice through microCT and histologic and immunohistochemical analysis. MATERIALS AND METHODS: A group of 30 male 129/Sv mice was equally subdivided into two groups: (1) nondiabetic (ND), in which mice did not undergo systemic alterations and received a standard diet, and (2) diabetic (D), in which mice were provided a high-fat diet from the age of 6 weeks until the conclusion of the study and received two intraperitoneal (IP) injections of streptozotocin (STZ) at a concentration of 100 mg/Kg each. Each mouse underwent extraction of a maxillary first molar, and customized Ti screws (0.50 mm diameter, 1.5 mm length) were placed in the residual alveolar sockets of the palatal roots. At 7 and 21 days after implant placement, the animals were euthanized for maxilla and pancreas collection. Maxillae containing Ti implants were analyzed with microCT, histology, and immunohistochemistry for cells that were positive for F4/80, CD146, runt-related transcription factor 2 (Runx2), and proliferating cell nuclear antigen (PCNA). Pancreata were histologically analyzed. Quantitative data were statistically analyzed with a significance level at 5% (P < .05). RESULTS: ND mice presented successful healing and osseointegration, with a significantly higher fraction of bone volume compared to D mice, both at the alveolar sockets (53.39 ± 5.93 and 46.08 ± 3.18, respectively) and at the implant sites (68.88 ± 7.07 and 44.40 ± 6.98, respectively) 21 days after implant placement. Histologic evaluation revealed that the ND mice showed a significant decrease in inflammatory infiltrate and a significant increase in newly formed bone matrix at 21 days, whereas peri-implant sites in the D mice were predominantly encapsulated by fibrous tissue and chronic inflammatory infiltrate. Immunohistochemical characterization revealed higher Runx2 osteoblast differentiation and higher cell proliferation activity in the ND mice at 7 days, while higher amounts of macrophages were present in D mice at 7 and 21 days. Interestingly, no differences were found in CD146-positive cells when comparing ND and D mice. CONCLUSIONS: This study evaluated the effects of immediate dental implant placement in 129/Sv diabetic mice by using specific healing markers to identify changes in cellular events involved in early oral osseointegration. This approach may serve as tool to evaluate new materials and surface coatings to improve osseointegration in diabetic patients.

Revolutionizing fracture fixation in diabetic and non-diabetic rats: High mobility group box 1-based coating for enhanced osseointegration.

Chronic inflammation and hyperglycemia in diabetic patients increase the risk of implant failure and impaired fracture healing. We previously developed and characterized a titanium (Ti) coating strategy using an imidazolium-based ionic liquid (IonL) with a fully reduced, non-oxidizable High Mobility Group Box 1 (HMGB1) isoform (Ti-IonL-HMGB1) to immunomodulate tissue healing. In this study, we used an open reduction fracture fixation (ORIF) model in non-diabetic (ND) and diabetic (D) rats to further investigate the effectiveness of this Ti-IonL-HMGB1 coating on orthopedic applications. Ninety male Lewis rats (12-15 weeks) were divided into D (n = 45) and ND (n = 45) groups that were distributed into three subgroups based on the type of local treatment received: Ti (uncoated Ti), Ti-IonL, and Ti-IonL-HMGB1 implants. Fracture healing and osseointegration were evaluated using microtomographic, histological, and immunohistochemical analysis of proliferating cell nuclear antigen (PCNA), Runt-related transcription factor 2 (RUNX2), and HMGB1 markers at 2, 10, and 21 days post-ORIF. Scanning Electron Microscopy verified the coating stability after placement. Microtomographic and histological analysis demonstrated increased fracture healing and osseointegration for ND rats in all treatment groups at 10 days, with impaired healing for D rats. Immunohistochemical analysis exhibited elevated PCNA+ and RUNX2+ cells for D animals treated with Ti-IonL-HMGB1 at 21 days compared to all other groups. The immunohistochemical marker HMGB1 was elevated at all time points for D animals in comparison to ND animals, yet was lowered for D tissues near the Ti-IonL-HMGB1 treated implant. Improved osseous healing was demonstrated in D animals with Ti-IonL-HMGB1 treatment by 21 days, compared to D animals with other treatments. To the best of our knowledge, this is the first study analyzing Ti-IonL-HMGB1 implantation in an injury site through ORIF procedures in ND and D rats. This surface approach has potential for improving implanted biomaterials in diabetic environments.

Biological Effects of New Titanium Surface Coatings Based on Ionic Liquids and HMGB1: A Cellular and Molecular Characterization in Lewis Rats.

High Mobility Group Box 1 (HMGB1) is a redox-sensitive molecule that plays dual roles in tissue healing and inflammation. We previously demonstrated that HMGB1 is stable when anchored by a well-characterized imidazolium-based ionic liquid (IonL), which serves as a delivery vehicle for exogenous HMGB1 to the site of injury and prevents denaturation from surface adherence. However, HMGB1 exists in different isoforms [fully reduced HMGB1 (FR), a recombinant version of FR resistant to oxidation (3S), disulfide HMGB1 (DS), and inactive sulfonyl HMGB1(SO)] that have distinct biological functions in health and disease. Thus, the goal of this study was to evaluate the effects of different recombinant HMGB1 isoforms on the host response using a rat subcutaneous implantation model. A total of 12 male Lewis rats (12-15 weeks) were implanted with titanium discs containing different treatments (n = 3/time point; Ti, Ti-IonL, Ti-IonL-DS, Ti-IonL-FR, and Ti-IonL-3S) and assessed at 2 and 14 days. Histological (H&E and Goldner trichrome staining), immunohistochemistry, and molecular analyses (qPCR) of surrounding implant tissues were employed for analysis of inflammatory cells, HMGB1 receptors, and healing markers. Ti-IonL-DS samples resulted in the thickest capsule formation, increased pro-inflammatory, and decreased anti-inflammatory cells, while Ti-IonL-3S samples demonstrated suitable tissue healing similar to uncoated Ti discs, as well as an upregulation of anti-inflammatory cells at 14 days compared to all other treatments. Thus, results from this study demonstrated that Ti-IonL-3S are safe alternatives for Ti biomaterials. Future studies are necessary to investigate the healing potential of Ti-IonL-3S in osseointegration scenarios.

Influence of age and gender on alveolar bone healing post tooth extraction in 129 Sv mice: a microtomographic, histological, and biochemical characterization.

OBJECTIVES: To analyze the effect of biological sex and aging on craniofacial bone features in 129 Sv mice and their influence on dental socket healing post tooth extraction. MATERIALS AND METHODS: A total of 52 129 Sv mice were used, of which 28 were young (3-4 months) and 24 were aged (17-18 months), equally distributed according to biological sex. After an upper right incisor extraction, mice specimens were collected at 7, 14, and 21-days post-surgery for microtomographic (microCT) and comprehensive histological analysis. Mandible, skull bones, and maxillae at 21 days were analyzed by microCT, while blood plasma samples were collected for the detection of key bone turnover markers (P1NP and CTX-1) by enzyme-linked immunosorbent (ELISA) assay. RESULTS: Aged females depicted significantly decreased mineralized bone content in alveolar sockets in comparison to young females and aged males at day 7, and aged males at day 14. Mandible RCA and Ma.AR of aged females were also significantly decreased in comparison with young females. Histological evaluation revealed that all alveolar sockets healed at 21 days with inflammation resolution and deposition of new bone. Immunohistochemistry for TRAP revealed increased area density for osteoclasts in alveolar sockets of aged females when compared to young females at 21 days. While a significant increase in CTX-1 levels was detected in blood plasma of aged females when compared to young females, P1NP levels did not significantly change between young and older females. No significant changes were observed for males. CONCLUSIONS: Age and gender can significantly affect craniofacial bones of 129 Sv mice, especially maxilla and mandible in females. Considering the altered bone resorption parameters and delayed alveolar bone healing in older females, careful deliberation is necessary during development of pre-clinical models for craniofacial research. CLINICAL RELEVANCE: Aging can be a contributing factor to slower bone healing in craniofacial bones. However, there are no sufficient experimental studies that have addressed this phenomenon along with biological sex taken into consideration.

A Model Study to Evaluate Osseointegration and Fracture Healing Following Open Reduction and Internal Fixation (ORIF) in Diabetic Lewis Rats.

There is a higher risk of implant osseointegration failure after open reduction and internal fixation (ORIF) in patients with diabetes due to increased inflammatory conditions, associated metallic corrosion and infection. While it is possible to avoid elective osseous surgery in patients with diabetes, it may not be the case in nonelective cases, such as ORIF ankle fractures. A total of 30 male Lewis rats (12-15 weeks old) were distributed into diabetic (D) and nondiabetic (ND) groups. Fracture healing and osseointegration were evaluated at 2-, 10-, and 21-day time points. Microtomographic and histological analysis depicted distinct differences in fracture healing and osseointegration between D and ND animals. Immunohistochemical analysis exhibited elevated proliferation (PCNA) and osteogenic (Runx2) cells for ND animals, while HMGB1 (inflammatory marker) was elevated for D animals during healing. Bone resorption marker CTX-1 was elevated in the plasma of D animals at 2 days, while bone formation marker P1NP was higher for ND animals at 10 days. Overall, this model resulted in delayed implant osseointegration and fracture healing in diabetic animals, highlighting the importance of developing new biomaterials or implant coatings that can improve bone healing outcomes in this patient population.

Effects of Dicationic Imidazolium-Based Ionic Liquid Coatings on Oral Osseointegration of Titanium Implants: A Biocompatibility Study in Multiple Rat Demographics.

Dicationic imidazolium-based ionic liquids with amino acid anions, such as IonL-phenylalanine (IonL-Phe), have been proposed as a multifunctional coating for titanium (Ti) dental implants. However, there has been no evaluation of the biocompatibility of these Ti coatings in the oral environment. This study aims to evaluate the effects of IonL-Phe on early healing and osseointegration of Ti in multiple rat demographics. IonL-Phe-coated and uncoated Ti screws were implanted into four demographic groups of rats to represent biological variations that could affect healing: young males (YMs) and females (YFs), ovariectomized (OVXFs) females, and old males (OMs). Samples underwent histopathological and histomorphometric analysis to evaluate healing at 7 and 30 days around IonL-coated and uncoated Ti. The real-time quantitative polymerase chain reaction was also conducted at the 2- and 7-day YM groups to evaluate molecular dynamics of healing while the IonL-Phe was present on the surface. IonL-coated and uncoated implants demonstrated similar histological signs of healing, while coated samples' differential gene expression of immunological and bone markers was compared with uncoated implants at 2 and 7 days in YMs. While YMs presented suitable osseointegration for both uncoated and IonL-Phe-coated groups, decreased success rate in other demographics resulted from lack of supporting bone in YFs and poor bone quality in OVXFs and OMs. Overall, it was found that IonL-coated samples had increased bone-to-implant contact across all demographic groups. IonL-Phe coating led to successful osseointegration across all animal demographics and presented the potential to prevent failures in scenarios known to be challenged by bacteria.

Succession of oral bacterial colonizers on dental implant materials: An in vitro biofilm model.

OBJECTIVES: Oral bacterial adhesion on dental implant materials has been extensively studied using in vitro systems but has yielded results restricted to in vitro growth patterns due to limitations in species selection, sustained fastidious anaerobe growth, and mixed culture longevity. The aim of this study was to develop an oral bacterial biofilm model consisting of colonizers representative of the oral microbiome exhibiting temporal shifts characteristic of plaque development and maturation in vivo. METHODS: Streptococcus oralis, Actinomyces naeslundii, Aggregatibacter actinomycetemcomitans, Veillonella parvula, Fusobacterium nucleatum, and Porphyromonas gingivalis were grown in monoculture prior to combination in mixed culture. Commercially pure titanium (cpTi) and yttria-stabilized zirconia (ZrO2) disks with polished, acid-etched, or sandblasted surfaces were prepared to evaluate oral bacterial adhesion. After 6 h, 1, 3, 7, 14 and 21 days, genomic DNA from planktonic and adherent bacteria was isolated. Quantitative polymerase chain reaction (qPCR) was used to enumerate the amount and proportion of each species. RESULTS: Early-colonizing S. oralis and A. actinomycetemcomitans, dominated after 6 h prior to secondary colonization by F. nucleatum and V. parvula in planktonic (1 day) and sessile (3 days) form. A. naeslundii maintained relatively low but stable bacterial counts throughout testing. After 14 days, late-colonizing P. gingivalis became established in mixed culture and persisted, becoming the dominant species after 21 days. The composition of adherent bacteria across all substrates was statistically similar at all timepoints with notable exceptions including lower S. oralis bacterial counts on polished cpTi (3 days). SIGNIFICANCE: Within the present model's limitations, multispecies oral bacterial attachment is similar on surface-treated cpTi and ZrO2.

Molecular-Level Understanding of the Influence of Ions and Water on HMGB1 Adsorption Induced by Surface Hydroxylation of Titanium Implants.

Due to its excellent chemical and mechanical properties, titanium has become the material of choice for orthopedic and dental implants to promote rehabilitation via bone anchorage and osseointegration. Titanium osseointegration is partially related to its capability to form a TiO2 surface layer and its ability to interact with key endogenous proteins immediately upon implantation, establishing the first bone-biomaterial interface. Surgical trauma caused by implantation results in the release of high-mobility group box 1 (HMGB1) protein, which is a prototypic DAMP (damage-associated molecular pattern) with multiple roles in inflammation and tissue healing. To develop different surface strategies that improve the clinical outcome of titanium-based implants by controlling their biological activity, a molecular-scale understanding of HMGB1-surface interactions is desired. Here, we use molecular dynamics (MD) computer simulations to provide direct insight into the HMGB1 interactions and the possible molecular arrangements of HMGB1 on fully hydroxylated and nonhydroxylated rutile (110) TiO2 surfaces. The results establish that HMGB1 is most likely to be adsorbed directly onto the surface regardless of surface hydroxylation, which is undesirable because it could affect its biological activity by causing structural changes to the protein. The hydroxylated TiO2 surface shows a greater affinity for HMGB1 than the nonhydroxylated surface. The water layer on the nonhydroxylated TiO2 surface prevents ions and the protein from directly contacting the surface. However, it was observed that if the ionic strength increases, the total number of ions adsorbed on the two surfaces increases and the protein's direct adsorption ability decreases. These findings will help to understand the HMGB1-TiO2 interactions upon implantation as well as the development of different surface strategies by introducing ions or ionic materials to the titanium implant surface to modulate its interactions with HMGB1 to preserve biological function.

Diabetes as a Risk Factor for Orthopedic Implant Surface Performance: A Retrieval and In Vitro Study.

Orthopedic devices are often associated with increased risk for diabetic patients due to impaired wound healing capabilities. Adverse biological responses for immunocompromised patients at the implant-tissue interface can lead to significant bone resorption that may increase failure rates. The goal of this study was to characterize the surface of implants removed from diabetic patients to determine underlying mechanisms of diabetes-induced impaired osseointegration. Thirty-nine retrieved titanium and stainless-steel orthopedic devices were obtained from diabetic and non-diabetic patients, and compared to non-implanted controls. Optical Microscopy, Scanning Electron Microscopy, Energy Dispersive X-ray Spectroscopy, and X-ray Photoelectron Spectroscopy revealed changes in morphology, chemical composition, oxidation state, and oxide thickness of the retrieval specimens, respectively. Additionally, titanium disks were immersed for 28 days in simulated in vitro diabetic conditions followed by Inductively Coupled Plasma-Optical Emission Spectroscopy to quantify metal dissolution. Electrochemical testing was performed on specimens from retrievals and in vitro study. Aside from biological deposits, retrievals demonstrated surface discoloration, pit-like formations and oxide thinning when compared to non-implanted controls, suggesting exposure to unfavorable acidic conditions. Cyclic load bearing areas on fracture-fixation screws and plates depicted cracking and delamination. The corrosion behavior was not significantly different between diabetic and non-diabetic conditions of immersed disks or implant type. However, simulated diabetic conditions elevated aluminum release. This elucidates orthopedic implant failures that potentially arise from diabetic environments at the implant-tissue interface. Design of new implant surfaces should consider specific strategies to induce constructive healing responses in immunocompromised patients while also mitigating corrosion in acidic diabetic environments.

Cellular and Molecular Dynamics during Early Oral Osseointegration: A Comprehensive Characterization in the Lewis Rat.

OBJECTIVE: There is a need to improve the predictability of osseointegration in implant dentistry. Current literature uses a variety of in vivo titanium (Ti) implantation models to investigate failure modes and test new materials and surfaces. However, these models produce a variety of results, making comparison across studies difficult. The purpose of this study is to validate an oral osseointegration in the Lewis rat to provide a reproducible baseline to track the inflammatory response and healing of Ti implants. METHODS: Ti screws (0.76 mm Ø × 2 mm length) were implanted into the maxillary diastema of 52 adult male Lewis rats. Peri-implant tissues were evaluated 2, 7, 14, and 30 days after implantation (n = 13). Seven of the 13 samples underwent microtomographic analysis, histology, histomorphometry, and immunohistochemistry to track healing parameters. The remaining six samples underwent quantitative polymerase chain reaction (qPCR) to evaluate gene expression of inflammation and bone remodeling markers over time. RESULTS: This model achieved a 78.5% success rate. Successful implants had a bone to implant contact (BIC)% of 68.86 ± 3.15 at 30 days on average. Histologically, healing was similar to other rodent models: hematoma and acute inflammation at 2 days, initial bone formation at 7, advanced bone formation and remodeling at 14, and bone maturation at 30. qPCR indicated the highest expression of bone remodeling and inflammatory markers 2-7 days, before slowly declining to nonsurgery control levels at 14-30 days. CONCLUSION: This model combines cost-effectiveness and simplicity of a rodent model, while maximizing BIC, making it an excellent candidate for evaluation of new surfaces.

Mammalian cell response and bacterial adhesion on titanium healing abutments: effect of multiple implantation and sterilization cycles.

OBJECTIVE: Multiple implantations of the implant healing abutment (IHA) could adversely impact its surface properties in vivo. Furthermore, the effect of sterilization and reuse of the IHA on soft tissue viability and bacterial contamination has not been extensively studied. The goal of this study was to perform an in vitro analysis of mammalian cell viability and bacterial adhesion on the surfaces of retrieved IHA after single and multiple implantations and repetitive cycles of sterilization. MATERIALS AND METHODS: IHA surface morphology was studied using optical microscopy. Cell viability of gingival fibroblasts (HGF-1) and oral keratinocytes (HOKg) in indirect contact with IHAs was assessed for 3 and 7 days. Immersion in bacterial culture was performed with a polyculture of Streptococcus species for 3 days and Streptococcus species with Fusobacterium nucleatum for 7 days. RESULTS: IHAs exhibited signs of surface damage even after a single exposure to the oral cavity. Fibroblasts did not show a significant preference towards control IHAs over used IHAs, whereas keratinocytes exhibited a significant decrease in viability when exposed to IHAs after multiple implantation cycles as compared with controls. Adherent bacterial count increased with increasing number of IHA implantations for both polycultures. CONCLUSIONS: Reusing of IHAs in vivo promoted surface degradation in addition to adversely impacting host cell viability and oral bacterial attachment in vitro. These findings show IHA reuse might potentially affect its clinical performance. CLINICAL RELEVANCE: Careful consideration should be taken when reusing IHAs in patients because this practice can result in permanent surface changes that might affect soft tissue integration during the healing period and promote bacterial colonization.

Investigation of the early healing response to dicationic imidazolium-based ionic liquids: a biocompatible coating for titanium implants.

Dicationic Imidazolum-based ionic liquids with amino acid anions (IonL) have been proposed as a multifunctional coating for titanium dental implants, as their properties have been shown to address multiple early complicating factors while maintaining host cell compatibility. This study aims to evaluate effects of this coating on host response in the absence of complicating oral factors during the early healing period using a subcutaneous implantation model in the rat. IonLs with the best cytocompatibility and antimicrobial properties (IonL-Phe, IonL-Met) were chosen as coatings. Three different doses were applied to cpTi disks and subcutaneously implanted into 36 male Lewis rats. Rats received 2 implants: 1 coated implant on one side and an uncoated implant on the contralateral sides (n=3 per formulation, per dose). Peri-implant tissue was evaluated 2 and 14 days after implantation with H&E staining and IHC markers associated with macrophage polarization as well as molecular analysis (qPCR) for inflammatory and healing markers. H&E stains revealed the presence of the coating, blood clots and inflammatory infiltrate at 2 days around all implants. At 14 days, inflammation had receded with more developed connective tissue with fibroblasts, blood vessels in certain doses of coated and uncoated samples with no foreign body giant cells. This study demonstrated that IonL at the appropriate concentration does not significantly interfere with and healing and Ti foreign body response. Results regarding optimal dose and formulation from this study will be applied in future studies using an oral osseointegration model.

Effects of multiple implantations of titanium healing abutments: Surface characteristics and microbial colonization.

OBJECTIVE: Very few studies have investigated dental implant components involved in the early stage of healing, especially the implant healing abutment (IHA), despite its vital role in soft tissue contouring and shaping after implant placement. Although these components are labelled by the manufacturer for "single-use only," it is a common clinical practice to clean, sterilize, and reuse them. METHODS: In the present study, IHAs after single and multiple implantations were retrieved as per standard procedures, and biological material isolated from the surface was subjected to 16S rRNA sequence analysis. The microbiome analysis was followed by cleaning and sterilization in order to replicate clinical sterilization techniques. Following sterilization, retrievals were subjected to surface characterization with optical and scanning electron microscopy to investigate surface features, and electrochemical testing was performed to evaluate corrosion behavior. RESULTS: The microbiota was comprised of early colonizers including Streptococcus species and secondary anaerobic colonizers such as Fusobacterium, Capnocytophaga, and Prevotella species. The surface analysis revealed that irrespective of the cleaning and sterilization techniques, the pristine, homogeneous surface of the new, unused IHAs could not be restored. Both single and multiple-use IHAs had severe surface changes including discoloration, major abrasions, biological contamination, and the IHA retrievals exhibited higher corrosion rate as compared to control specimens. SIGNIFICANCE: Reusing IHAs multiple times may not be a prudent practice as the microbial colonization and surface changes caused by using this component multiple times may affect the performance of IHAs in soft tissue healing.

HGMB1 and RAGE as Essential Components of Ti Osseointegration Process in Mice.

The release of the prototypic DAMP High Mobility Group Box 1 (HMGB1) into extracellular environment and its binding to the Receptor for Advanced Glycation End Products (RAGE) has been described to trigger sterile inflammation and regulate healing outcome. However, their role on host response to Ti-based biomaterials and in the subsequent osseointegration remains unexplored. In this study, HMGB1 and RAGE inhibition in the Ti-mediated osseointegration were investigated in C57Bl/6 mice. C57Bl/6 mice received a Ti-device implantation (Ti-screw in the edentulous alveolar crest and a Ti-disc in the subcutaneous tissue) and were evaluated by microscopic (microCT [bone] and histology [bone and subcutaneous]) and molecular methods (ELISA, PCR array) during 3, 7, 14, and 21 days. Mice were divided into 4 groups: Control (no treatment); GZA (IP injection of Glycyrrhizic Acid for HMGB1 inhibition, 4 mg/Kg/day); RAP (IP injection of RAGE Antagonistic Peptide, 4 mg/Kg/day), and vehicle controls (1.5% DMSO solution for GZA and 0.9% saline solution for RAP); treatments were given at all experimental time points, starting 1 day before surgeries. HMGB1 was detected in the Ti-implantation sites, adsorbed to the screws/discs. In Control and vehicle groups, osseointegration was characterized by a slight inflammatory response at early time points, followed by a gradual bone apposition and matrix maturation at late time points. The inhibition of HMGB1 or RAGE impaired the osseointegration, affecting the dynamics of mineralized and organic bone matrix, and resulting in a foreign body reaction, with persistence of macrophages, necrotic bone, and foreign body giant cells until later time points. While Control samples were characterized by a balance between M1 and M2-type response in bone and subcutaneous sites of implantation, and also MSC markers, the inhibition of HMGB1 or RAGE caused a higher expression M1 markers and pro-inflammatory cytokines, as well chemokines and receptors for macrophage migration until later time points. In conclusion, HMGB1 and RAGE have a marked role in the osseointegration, evidenced by their influence on host inflammatory immune response, which includes macrophages migration and M1/M2 response, MSC markers expression, which collectively modulate bone matrix deposition and osseointegration outcome.

The role of bacterial biofilm and mechanical forces in modulating dental implant failures.

Currently many assume that bacteria are the primary etiological factor associated with failure of titanium dental implants. However, emerging data indicates a possible role for mechanical forces in implant failure. This study is based on the hypothesis that the synergistic effect of mechanical forces and bacterial biofilm can lead to surface damage resulting in in vivo release of metallic particles. The primary aim of the study was to develop a dynamic fatigue test method for dental implants immersed in wet environments such as; (i) 0.01 M phosphate buffer saline (PBS); (ii) lactic acid (pH = 5); (iii) bacterial polyculture. Four dental implants each were subjected to fatigue loading from 45 N to 450 N at 4 Hz for 2 million cycles while immersed in (i) PBS (negative control); (ii) bacterial culture (test); and (iii) lactic acid (positive control). Post-testing, optical microscopy, x-ray photoelectron spectroscopy, and electrochemical corrosion tests were performed to evaluate the surface morphology, chemistry, and potential, respectively, of titanium implants. Post-testing, surface discoloration was evident in all three groups. However, the surface damage was further established in XPS analyses of test specimens, which showed that the interplay of bacterial biofilm and mechanical forces resulted in thinning of the TiO2. Lower corrosion potential (Ecorr) of the test specimens compared to positive and negative controls also illustrated damage to the oxide layer. However, other electrochemical parameters such as linear polarization resistance (LPR) and corrosion rate (CR) were comparable among the groups indicating the corrosion resistance post-testing. The synergistic effect of cyclic occlusal loading and bacteria biofilm could negatively affect the surface of titanium dental implants.

Detection of apoptotic and live pre-osteoblast cell line using impedance-based biosensors with variable electrode design.

Electrical impedance-based sensing of cell activity has become a powerful analytical tool that allows the monitoring of several relevant biological processes associated with cell evolution and morphology. In these types of biosensors, the electrode design has a direct impact on the sensitivity because it defines the capability of the biosensor to measure small changes in the impedance resulting from cell activities. Herein, impedance-based biosensors arrays with several configurations were successfully developed and used to study the impact of the electrode layout on the dynamics of cultured pre-osteoblast cells. The biosensor design was initially validated by measuring the effect of electrode design on the capacitance of a dielectric polymer (parylene) that mimics the dielectric characteristics of cell populations, results are shown in the Supplementary information section. Results from in vitro cell growth indicate that the optimized design of single electrodes with a diameter of 50 µm, are the most sensitive to cell motion whereas increasing the number of electrodes allows clear differentiation between living and dead cells after 3 h of inducing apoptosis. Apoptosis death was induced with Staurosporine, a chemical mediator of apoptosis in osteoblasts. These impedance results have been validated with optical imaging and flow cytometry analysis that were performed on parallel cultures. Frequency and electrolyte concentration effects are also discussed.

In Vitro Evaluation of Cell Compatibility of Dental Cements Used with Titanium Implant Components.

PURPOSE: To evaluate the biocompatibility of five dental cement compositions after directly exposing human gingival fibroblast (HGF) and MC3T3-E1 preosteoblast cells to cement alone and cement applied on commercially pure titanium (cpTi) specimens. MATERIALS AND METHODS: Nanostructurally integrated bioceramic (NIB), resin (R), resin-modified glass ionomer (RMGIC), zinc oxide eugenol (ZOE), and zinc phosphate (ZP) compositions were prepared according to the respective manufacturer's instructions. Samples were prepared in cylindrical Teflon molds or applied over the entire surface of polished cpTi discs. All samples were cured for 0.5, 1, 12, or 24 hours post-mixing. Direct contact testing was conducted according to ISO 10993 by seeding 6-well plates at 350,000 cells/well. Plates were incubated at 37°C in a humidified atmosphere with 5% CO2 for 24 hours before individually plating samples and cpTi control discs. Plates were then incubated for an additional 24 hours. Microtetrazolium (MTT) cell viability assays were used to measure sample cytotoxicity. RESULTS: For samples that cured for 24 hours prior to direct contact exposure, only NIB and ZP cements when cemented on cpTi demonstrated cell viability percentages above the minimum biocompatibility requirement (≥70%) for both the investigative cell lines. R, RMGIC, and ZOE cements exhibited moderate to severe cytotoxic effects on both cell lines in direct contact and when cemented on cpTi specimens. For HGF cells, ZOE cemented-cpTi specimens exhibited significantly decreased cytotoxicity, whereas RMGIC cemented-cpTi specimens exhibited significantly increased cytotoxicity. CONCLUSIONS: Despite previous studies that showed enhanced cpTi corrosion activity for fluoride-containing compositions (NIB and ZP), there was no significant difference in cytotoxicity between cement alone and cemented-cpTi. In general, the MC3T3-E1 preosteoblast cells were more sensitive than HGF cells to cement composition. Ultimately, cement composition played a significant role in maintaining host cell compatibility. Results of this work help illustrate the impact of different cement formulations on host cell health and emphasize the need for understanding material properties when selecting certain formulations of dental cements, which can ultimately influence the survival of dental implant systems.

Evaluation of oral microbial corrosion on the surface degradation of dental implant materials.

BACKGROUND: Titanium (Ti) dominates as the material of choice for dental implant systems. Recently, titanium-zirconium alloy (TiZr) and zirconia (ZrO2 ) have emerged as alternative materials due to higher mechanical strength and lower corrosion susceptibility. Oral pathogenic bacteria can colonize Ti surfaces, leading to surface degradation, which has yet to be investigated on TiZr and ZrO2 . The aim of this study was to compare in vitro oral bacterial adhesion and subsequent surface degradation on commercial Ti, TiZr, and ZrO2 implants. METHODS: Ti, TiZr, and ZrO2 implants with sandblasted, acid-etched (SLA) surfaces in addition to modified SLA-treated (modSLA) Ti implants (n = 3) were immersed for 30 consecutive days in Streptococcus polyculture. Post-immersion, adherent bacterial count was quantified. Optical microscopy was used to assess qualitative degradation and score Ti-based implants based on degree of surface damage while electrochemical testing quantified corrosion behavior. Analysis of variance followed by post-hoc Tukey test was used to statistically compare quantitative results (α = 0.05). RESULTS: Ti-SLA, Ti-modSLA, and TiZr-SLA implants exhibited localized features characteristic of corrosion attack while ZrO2 -SLA implants experienced minimal changes in surface morphology as compared to non-immersed control. Corrosion features were more numerous on Ti-modSLA implants but smaller in size as compared with those on Ti-SLA and TiZr-SLA implants. No significant differences in corrosion resistance (polarization resistance and corrosion rate) were observed between Ti-SLA, Ti-modSLA, and TiZr-SLA implants. CONCLUSION: TiZr and ZrO2 dental implant surfaces were not more susceptible to colonization and surface degradation by oral Streptococcus species than commercially pure Ti implants.