Comparison accuracy of digital and analog method using milled occlusal splints.

OBJECTIVE: This clinical trial aims to compare the accuracy of interocclusal registration in centric relation taken with polyvinyl siloxane and intraoral scanner (IOS) with the fabrication of occlusal devices. METHODS: Thirty-one participants were included in the trial registered at ClinicalTrials.gov (NCT05317182) receiving two different occlusal devices from two different workflows. One workflow was performed using polyvinyl siloxane impression material and the other with IOS. Every splint was scanned before and after the occlusal adjustments to compare the volumetric changes using the Root Mean Square deviation (RMS). Furthermore, three evaluators assessed the 3D comparison using color maps in a Visual Analog Scale (VAS). RESULTS: The average values of RMS were higher for the analog approach (0.01 ± 0.067) than the digital approach (0.065 ± 0.035). However, the differences were not statistically significant (p < 0.063) between the two impression techniques. For the semiquantitative analysis performed by blinded evaluators, differences in VAS values between the impression for the digital (2.08 ± 2.4) and analog (3.80 ± 3.3) technique were statistically significant. The three evaluators agreed in more than 90% of the qualitative dichotomous evaluation. CONCLUSION: Digital impressions did not show inferior accuracy compared to conventional impressions when assessed using quantitative measurements. CLINICAL SIGNIFICANCE: This clinical trial provided evidence on registering interocclusal relationship at increased vertical dimension with fully digital workflow for complete arch prosthesis.

CAD-CAM and analog occlusal splints comparison based on the amount of occlusal adjustments. 3D analysis of the volumetric changes: A pilot study.

OBJECTIVE: To evaluate the volumetric changes on occlusal surface of computer-aided design and computer-aided manufacturing (CAD-CAM) occlusal devices fabricated following a fully digital workflow after occlusal adjustment, compared to those fabricated with an analog workflow. MATERIALS AND METHODS: Eight participants were included in this clinical pilot study, receiving two different occlusal devices fabricated with two different workflows, fully analog and fully digital. Every occlusal device was scanned before and after the occlusal adjustments to compare the volumetric changes using a reverse engineering software program. Moreover, three independent evaluators assessed a semi-quantitative and qualitative comparison using visual analog scale and dichotomous evaluation. The Shapiro-Wilk test was performed to validate normal distribution assumption, and a dependent t-Student test for paired variables was used to determine statistically significant differences (p-value < 0.05). RESULTS: The root mean square value was extracted from the 3-Dimensional (3D) analysis of the occlusal devices. The average values of the root mean square were higher for the analogic technique (0.23 ± 0.10 mm) than the digital technique (0.14 ± 0.07 mm) but the differences were not statistically significant (paired t-Student test; p = 0.106) between the two fabrication techniques. The semiquantitative visual analog scale values between the impression for the digital (5.08 ± 2.4 cm) and analog (3.80 ± 3.3 cm) technique were significant (p < 0.001), and statistically significant differences values were assessed for evaluator 3 compared to the other evaluators (p < 0.05). However, the three evaluators agreed on the qualitative dichotomous evaluation in 62% of the cases, and at least two evaluators agreed in 100% of the evaluations. CONCLUSIONS: Occlusal devices fabricated following a fully digital workflow resulted in fewer occlusal adjustments, as they could be a valid alternative to those fabricated following an analog workflow. CLINICAL SIGNIFICANCE: Fabricated occlusal devices following a fully digital workflow could have some advantages over analog workflow such reduce occlusal adjustments at delivery appointment, which can result in reduced chair time and therefore increased comfort for the patient and clinician.

Interactions of two enantiomers of a designer antimicrobial peptide with structural components of the bacterial cell envelope.

Antimicrobial peptides (AMPs) have great potential in treating multi-drug resistant bacterial infections. The antimicrobial activity of d-enantiomers is significantly higher than l-enantiomers and sometimes selectively enhanced against Gram-positive bacteria. Unlike phospholipids in the bacterial plasma membrane, the role of other bacterial cell envelop components is often overlooked in the mode of action of AMPs. In this work, we explored the structural interactions between the main different structural components in Gram-negative/Gram-positive bacteria and the two enantiomers of a designer AMP, GL13K. We observed that both l-GL13K and d-GL13K formed self-assembled amyloid-like nanofibrils when the peptides interacted with lipopolysaccharide and lipoteichoic acid, components of the outer membrane of Gram-negative bacteria and cell wall of Gram-positive bacteria, respectively. Another cell wall component, peptidoglycan, showed strong interactions exclusively with d-GL13K and formed distinct laminar structures. This specific interaction between peptidoglycans and d-GL13K might contribute to the enhanced activity of d-GL13K against Gram-positive bacteria as they have a much thicker peptidoglycan layer than Gram-negative bacteria. A better understanding of the specific role of bacterial cell envelop components in the AMPs mechanism of action can guide the design of more effective Gram-selective AMPs.

The parotid secretory protein BPIFA2 is a salivary surfactant that affects lipopolysaccharide action.

NEW FINDINGS: What is the central question of this study? The salivary protein BPIFA2 binds lipopolysaccharide, but its physiological function is not known. This study uses a new knockout mouse model to explore the physiological role of BPIFA2 in the oral cavity and systemic physiology. What is the main finding and its importance? BPIFA2 is a crucial surfactant in mouse saliva. In its absence, saliva exhibits the surface tension of water. Depletion of BPIFA2 affects salivary and ingested lipopolysaccharide and leads to systemic sequelae that include increased insulin secretion and metabolomic changes. These results suggest that the lipopolysaccharide-binding activity of BPIFA2 affects the activity of ingested lipopolysaccharide in the intestine and that BPIFA2 depletion causes mild metabolic endotoxaemia. ABSTRACT: Saliva plays important roles in the mastication, swallowing and digestion of food, speech and lubrication of the oral mucosa, antimicrobial and anti-inflammatory activities, and the control of body temperature in grooming animals. The salivary protein BPIFA [BPI fold containing family A member 2; former names: parotid secretory protein (PSP), SPLUN2 and C20orf70] is related to lipid-binding and lipopolysaccharide (LPS)-binding proteins expressed in the mucosa. Indeed, BPIFA2 binds LPS, but the physiological role of BPIFA2 remains to be determined. To address this question, Bpifa2 knockout (Bpifa2tm1(KOMP)Vlcg ) (KO) mice were phenotyped, with emphasis on the saliva and salivary glands. Stimulated whole saliva collected from KO mice was less able to spread on a hydrophobic surface than wild-type saliva, and the surface tension of KO saliva was close to that of water. These data suggest that BPIFA2 is a salivary surfactant that is mainly responsible for the low surface tension of mouse saliva. The reduced surfactant activity of KO saliva did not affect consumption of dry food or grooming, but saliva from KO mice contained less LPS than wild-type saliva. Indeed, mice lacking BPIFA2 responded to ingested LPS with an increased stool frequency, suggesting that BPIFA2 plays a role in the solubilization and activity of ingested LPS. Consistent with these findings, BPIFA2-depleted mice also showed increased insulin secretion and metabolomic changes that were consistent with a mild endotoxaemia. These results support the distal physiological function of a salivary protein and reinforce the connection between oral biology and systemic disease.

Dual Oral Tissue Adhesive Nanofiber Membranes for pH-Responsive Delivery of Antimicrobial Peptides.

Bioadhesive membranes with controllable and reversible underwater adhesion are desirable for several biomedical applications ranging from biosensing, drug/therapeutic delivery, and tissue regeneration. Here, we present dual soft mucosal and hard bone/enamel tissue adhesive nanofiber membranes composed of chitosan and pectin derivatives for pH-controlled delivery of antimicrobial peptides (AMPs) in the oral cavity. Ex vivo testing with porcine esophagus (soft mucosal mimic) indicated a 2-fold increase in the mucoadhesion of chitosan membranes with 0.05 wt % oxidized pectin coating, while the uncoated membranes exhibited 3-4-fold stronger adhesion to hydroxyapatite discs (enamel/hard bone mimic) compared to the coated membranes. The former is attributed to a synergistic interaction of surface nanofiber topography, intermolecular hydrogen bonding, and aldehyde-amine chemistry between surface polar groups and mucosal proteins, while the latter may arise from electrostatic interactions between cationic amines (-NH3+) in chitosan and anionic phosphates (-PO43-) in hydroxyapatite. Further, the dual hard-soft oral tissue adhesive nanofiber membranes loaded with cationic amphipathic AMPs (D-GL13K and IDR-1018) elicited pH-responsive AMP delivery and antimicrobial action comparable to chlorhexidine (CHX) against oral streptococci. Concurrently, the AMP loaded membranes were cytocompatible to both soft epithelial tissue-derived human oral keratinocytes and hard calvarial murine pre-osteoblast cells. We envision these membranes to function as adhesive gingival grafts and guided bone regeneration (GBR) membranes at the hard-soft tissue interface while simultaneously protecting against oral infections.

Dual Self-Assembled Nanostructures from Intrinsically Disordered Protein Polymers with LCST Behavior and Antimicrobial Peptides.

Antimicrobial peptides (AMPs) have attracted great interest as they constitute one of the most promising alternatives against drug-resistant infections. Their amphipathic nature not only provides them antimicrobial and immunomodulatory properties but also the ability to self-assemble into supramolecular nanostructures. Here, we propose their use as self-assembling domains to drive hierarchical organization of intrinsically disordered protein polymers (IDPPs). Using a modular approach, hybrid protein-engineered polymers were recombinantly produced, thus combining designer AMPs and a thermoresponsive IDPP, an elastin-like recombinamer (ELR). We exploited the ability of these AMPs and ELRs to self-assemble to develop supramolecular nanomaterials by way of a dual-assembly process. First, the AMPs trigger the formation of nanofibers; then, the thermoresponsiveness of the ELRs enables assembly into fibrillar aggregates. The interplay between the assembly of AMPs and ELRs provides an innovative molecular tool in the development of self-assembling nanosystems with potential use for biotechnological and biomedical applications.

Mechanism of fracture of NiTi superelastic endodontic rotary instruments.

The aim is to investigate the premature catastrophic fracture produced for different periods during clinical endodontic treatment of two brands of NiTi endodontic rotary instruments. 3 samples as-received, 6 samples used with patients for 2 and 7 h and 5 samples fractured were studied for each brand of endodontic NiTi rotary instruments. Transformation temperatures (Ms, Mf, As and Af) and enthalpies of transformation were determined by calorimetry. Critical stresses until fracture (σß→SIM, σSIM→ß) were obtained using an electromechanical testing machine. The samples were also visualized by Scanning Electron Microscopy. Calorimetric studies have shown an increase of the Ms and As transformation temperatures with time of use as well as a decrease of their stress transformations. Moreover, reverse transformation enthalpies decreased along the time. The enthalpies of transformation decreased because martensitic plates were anchored, which prevented their transformation to austenite; thus losing its superelastic effect. The stabilisation of the martensitic plates induced the collapse of the structure and so the main cause for the fracture. The heat treatment proposed has been increased the life in service of NiTi superelastic endodontic instruments recovering theirs superelastic effect.

In vitro cell response on CP-Ti surfaces functionalized with TGF-ß1 inhibitory peptides.

Osseointegration of implants is conversely related to the generation of a fibrous tissue capsule around the implant by the host environment. Although TGF-ß1 plays many roles in regeneration processes, it is the cytokine to be mostly associated to the production of fibrotic tissue and thus, its inhibition has demonstrated to be beneficial to prevent several fibrotic reactions. Surface biofunctionalization enables the immobilization of biologically active molecules on an implant surface to tailor the biological response of the host. Here, we studied in vitro biological effects of biofunctionalized CP-Ti surfaces with a TGF-ß1 inhibitor peptide, P144. A reliable biofunctionalization process that tethers P144 peptides to commercially pure titanium was developed. Differentiation of human mesenchymal stem cells, osteoblasts and fibroblasts on P144-functionalized and control surfaces was assessed at the gene expression and protein production levels. Results showed that P144-functionalized surfaces reduced expression and production of fibrotic differentiation markers and increased osteoblastic differentiation markers. Therefore, biofunctionalization of surfaces with TGF-ß1 inhibitor peptides are an alternative promising strategy for inducing osseointegration around medical devices and implants.

Solution and Solid-State Nuclear Magnetic Resonance Structural Investigations of the Antimicrobial Designer Peptide GL13K in Membranes.

The antimicrobial peptide GL13K encompasses 13 amino acid residues and has been designed and optimized from the salivary protein BPIFA2 to exhibit potent bacteriocidal and anti-biofilm activity against Gram-negative and Gram-positive bacteria as well as anti-lipopolysaccharide activity in vitro and in vivo. Here, the peptide was analyzed in a variety of membrane environments by circular dichroism spectroscopy and by high-resolution multidimensional solution nuclear magnetic resonance (NMR) spectroscopy. Whereas in the absence of membranes a random coil conformation predominates, the peptide adopts a helical structure from residue 5 to 11 in the presence of dodecylphosphocholine micelles. In contrast, a predominantly ß-sheet structure was observed in the presence of lipid bilayers carrying negatively charged phospholipids. Whereas 15N solid-state NMR spectra are indicative of a partial alignment of the peptide 15N-1H vector along the membrane surface, 2H and 31P solid-state NMR spectra indicate that in this configuration the peptide exhibits pronounced disordering activities on the phospholipid membrane, which is possibly related to antimicrobial action. GL13K, thus, undergoes a number of conformational transitions, including a random coil state in solution, a helical structure upon dilution at the surface of zwitterionic membranes, and ß-sheet conformations at high peptide:lipid ratios.

Chitosan-Recombinamer Layer-by-Layer Coatings for Multifunctional Implants.

The main clinical problems for dental implants are (1) formation of biofilm around the implant-a condition known as peri-implantitis and (2) inadequate bone formation around the implant-lack of osseointegration. Therefore, developing an implant to overcome these problems is of significant interest to the dental community. Chitosan has been reported to have good biocompatibility and anti-bacterial activity. An osseo-inductive recombinant elastin-like biopolymer (P-HAP), that contains a peptide derived from the protein statherin, has been reported to induce biomineralization and osteoblast differentiation. In this study, chitosan/P-HAP bi-layers were built on a titanium surface using a layer-by-layer (LbL) assembly technique. The difference in the water contact angle between consecutive layers, the representative peaks in diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), X-ray photoelectron spectroscopy (XPS), and the changes in the topography between surfaces with a different number of bi-layers observed using atomic force microscopy (AFM), all indicated the successful establishment of chitosan/P-HAP LbL assembly on the titanium surface. The LbL-modified surfaces showed increased biomineralization, an appropriate mouse pre-osteoblastic cell response, and significant anti-bacterial activity against Streptococcus gordonii, a primary colonizer of tissues in the oral environment.

Bioactive macroporous titanium implants highly interconnected.

Intervertebral implants should be designed with low load requirements, high friction coefficient and low elastic modulus in order to avoid the stress shielding effect on bone. Furthermore, the presence of a highly interconnected porous structure allows stimulating bone in-growth and enhancing implant-bone fixation. The aim of this study was to obtain bioactive porous titanium implants with highly interconnected pores with a total porosity of approximately 57 %. Porous Titanium implants were produced by powder sintering route using the space holder technique with a binder phase and were then evaluated in an in vivo study. The size of the interconnection diameter between the macropores was about 210 µm in order to guarantee bone in-growth through osteblastic cell penetration. Surface roughness and mechanical properties were analyzed. Stiffness was reduced as a result of the powder sintering technique which allowed the formation of a porous network. Compression and fatigue tests exhibited suitable properties in order to guarantee a proper compromise between mechanical properties and pore interconnectivity. Bioactivity treatment effect in novel sintered porous titanium materials was studied by thermo-chemical treatments and were compared with the same material that had undergone different bioactive treatments. Bioactive thermo-chemical treatment was confirmed by the presence of sodium titanates on the surface of the implants as well as inside the porous network. Raman spectroscopy results suggested that the identified titanate structures would enhance in vivo apatite formation by promoting ion exchange for the apatite formation process. In vivo results demonstrated that the bioactive titanium achieved over 75 % tissue colonization compared to the 40 % value for the untreated titanium.

Biomimetic treatment on dental implants for short-term bone regeneration.

OBJECTIVES: The main purpose of this work was to assess the short-term bone regenerative potential of new osteoconductive implants. The novelty of the study lies in the analysis of the effectiveness of a novel two-step treatment which combines shot-blasting with a thermo-chemical treatment, at very short times after implant placement in a minipig model. MATERIALS AND METHODS: Three hundred twenty implants with four different surface treatments, namely bioactivated surfaces, micro-rough grit-blasted, micro-rough acid-etched and smooth as-machined titanium implants were placed into the bone of 20 minipigs. The percent of bone-to-implant contact was determined 3 days, 1, 2, 3 and 10 weeks after implant placement by histomorphometric analysis. Surface composition, topography and wettability of the implant specimens were analysed. RESULTS: The combination of shot-blasting and thermo-chemical treatment accelerated bone regeneration at early stages in comparison with all other treatments between day 3 and week 3 (p < 0.05). The value of osseointegration attained at week 2 was maintained until the end of the experiment without any significant changes (percent direct contact ≈ 85 %). This was mostly attributed to the ability of these implants to form in vivo a layer of apatitic mineral that coated the implant and could rapidly stimulate bone nucleation and growth from the implant surface. CONCLUSIONS: The surface quality resulting from this treatment on cpTi provided dental implants with a unique ability of rapid bone regeneration and osseointegration. CLINICAL RELEVANCE: This treatment represents a step forward in the direction of reducing the time prior to implant loading.

The Effect of Implantoplasty on the Fatigue Behavior and Corrosion Resistance in Titanium Dental Implants.

Implantoplasty is a technique increasingly used to remove the biofilm that causes peri-implantitis on dental implants. This technique of mechanization of the titanium surface makes it possible to eliminate bacterial colonies, but it can generate variations in the properties of the implant. These variations, especially those in fatigue resistance and electrochemical corrosion behavior, have not been studied much. In this work, fatigue tests were performed on 60 dental implants without implantoplasty, namely 30 in air and 30 in Hank's solution at 37 °C, and 60 with implatoplasty, namely 30 in air and 30 in Hank's solution at 37 °C, using triaxial tension-compression and torsion stresses simulating human chewing. Mechanical tests were performed with a Bionix servo-hydraulic testing machine and fracture surfaces were studied by scanning electron microcopyElectrochemical corrosion tests were performed on 20 dental implants to determine the corrosion potentials and corrosion intensity for control implants and implantoplasty implants. Studies of titanium ion release to the physiological medium were carried out for each type of dental implants by Inductively Coupled-Plasma Mass Spectrometry at different immersion times at 37 °C. The results show a loss of fatigue caused by the implantoplasty of 30%, observing that the nucleation points of the cracks are in the areas of high deformation in the areas of the implant neck where the mechanization produced in the treatment of the implantoplasty causes an exaltation of fatigue cracks. It has been observed that tests performed in Hank's solution reduce the fatigue life due to the incorporation of hydrogen in the titanium causing the formation of hydrides that embrittle the dental implant. Likewise, the implantoplasty causes a reduction of the corrosion resistance with some pitting on the machined surface. Ion release analyses are slightly higher in the implantoplasted samples but do not show statistically significant differences. It has been observed that the physiological environment reduces the fatigue life of the implants due to the penetration of hydrogen into the titanium forming titanium hydrides which embrittle the implant. These results should be taken into account by clinicians to determine the convenience of performing a treatment such as implantoplasty that reduces the mechanical behavior and increases the chemical degradation of the titanium dental implant.

Characterisation of experimental flowable composites containing fluoride-doped calcium phosphates as promising remineralising materials.

OBJECTIVE: Remineralising composites with antibacterial properties may seal the cavity and prevent secondary caries. This study aimed at developing experimental flowable composites containing different concentrations of fluoride-doped calcium phosphate fillers and evaluating their remineralising and antibacterial properties. METHODS: Experimental resin-based composites containing different concentrations (0-20 %) of fluoride-doped calcium phosphate fillers (VS10/VS20) were formulated. The release of calcium (Ca), phosphate (PO) and fluoride (F) ions was assessed for 30 days. Remineralisation properties were evaluated through ATR-FTIR and SEM/EDX after storage in simulated body fluid (SBF). The metabolic activity and viability of Streptococcus gordonii was also evaluated through ATP, CFU and live/dead confocal microscopy. The evaluation of specific monomer elution from the experimental composites was conducted using high-performance liquid chromatography (HPLC). RESULTS: The composites containing VS10 showed the highest release of Ca, those containing VS20 released more F over time (p < 0.05), while there was no significant difference in terms of PO ions release between the groups (p > 0.05). A quick 7-day mineral precipitation was observed in the tested composites containing VS10 or VS20 at 10 %; these materials also showed the greatest antibacterial activity (p < 0.05). Moreover, the tested composites containing VS10 presented the lowest elution of monomers (p < 0.05). CONCLUSIONS: Innovative composites were developed with low monomers elution, evident antibacterial activity against S. gordonii and important remineralisation properties due to specific ions release. CLINICAL SIGNIFICANCE: Novel composites containing fluoride-doped calcium phosphates may be promising to modulate bacteria growth, promote remineralisation and reduce the risk of cytotoxicity related to monomers' elution.

Revision of ex vivo endodontic biofilm model using computer aided design.

OBJECTIVE: Most endodontic diseases are bacterium-mediated inflammatory or necrotic process induced by contaminated dental pulp. Although great advances are being performed to obtain more efficient antibacterial strategies for persistent infections, most studies lack of representative models to test their antibacterial effects and their outcomes cannot be promptly translated to clinical practice. Therefore, this study aimed to refine an ex vivo endodontic biofilm model combining human tooth, computer guided design and 3D printing to obtain a more reproducible and predictable model. METHODS: Monoradicular teeth were cut using three different methods: hand-held (HCC), mechanical precision (MPC) and computer aid guided cutting (CGC). Then, blocks were reassembled. The different model preparations were assessed in terms of dimensional tolerance, surface analysis, liquid tightness and Enterococcus faecalis biofilm development for 21 days, which was studied by metabolic assays and confocal microscopy. Then, the proposed model was validated using different commercial disinfecting treatments. RESULTS: CGC exhibited significantly lower deviation and surface without defects compared to HHC and MPC, leading to superior liquid tightness. Similarly, mature biofilms with high metabolic activity and vitality were observed in all conditions, CGC showing the lowest variation. Regarding the model validation, all antibacterial treatments resulted in the complete eradication of bacteria in the standard 2D model, whereas commercial treatments exhibited varying levels of efficacy in the proposed ex vivo model, from moderately reduction of metabolic activity to complete elimination of biofilm. CONCLUSIONS: The novel guided approach represents a more reliable, standardized, and reproducible model for the evaluation of endodontic disinfecting therapies. CLINICAL SIGNIFICANCE: During antibacterial treatment development, challenging 3D models using teeth substrates to test antibacterial treatments novel guided approach represents a more reliable, standardized, and reproducible model for the evaluation of endodontic disinfecting therapies.

Influence of Titanium Surface Residual Stresses on Osteoblastic Response and Bacteria Colonization.

Grit basting is the most common process applied to titanium dental implants to give them a roughness that favors bone colonization. There are numerous studies on the influence of roughness on osseointegration, but the influence of the compressive residual stress associated with this treatment on biological behavior has not been determined. For this purpose, four types of surfaces have been studied using 60 titanium discs: smooth, smooth with residual stress, rough without stress, and rough with residual stress. Roughness was studied by optic interferometry; wettability and surface energy (polar and dispersive components) by contact angle equipment using three solvents; and residual stresses by Bragg-Bentano X-ray diffraction. The adhesion and alkaline phosphatase (ALP) levels on the different surfaces were studied using Saos-2 osteoblastic cultures. The bacterial strains Streptococcus sanguinis and Lactobacillus salivarius were cultured on different surfaces, determining the adhesion. The results showed that residual stresses lead to increased hydrophilicity on the surfaces, as well as an increase in surface energy, especially on the polar component. From the culture results, higher adhesion and higher ALP levels were observed in the discs with residual stresses when compared between smooth and roughened discs. It was also found that roughness was the property that mostly influenced osteoblasts' response. Bacteria colonize rough surfaces better than smooth surfaces, but no changes are observed due to residual surface tension.

Interpenetrating nanofibrillar membrane of self-assembled collagen and antimicrobial peptides for enhanced bone regeneration.

Bone regeneration remains a major clinical challenge, especially when infection necessitates prolonged antibiotic treatment. This study presents a membrane composed of self-assembled and interpenetrating GL13K, an antimicrobial peptide (AMP) derived from a salivary protein, in a collagen membrane for antimicrobial activity and enhanced bone regeneration. Commercially available collagen membranes were immersed in GL13K solution, and self-assembly was initiated by raising the solution pH to synthesize the multifunctional membrane called COL-GL. COL-GL was composed of interpenetrating large collagen fibers and short GL13K nanofibrils, which increased hydrophobicity, reduced biodegradation from collagenase, and stiffened the matrix compared to control collagen membranes. Incorporation of GL13K led to antimicrobial and anti-fouling activity against early oral surface colonizer Streptococcus gordonii while not affecting fibroblast cytocompatibility or pre-osteoblast osteogenic differentiation. GL13K in solution also reduced macrophage inflammatory cytokine expression and increased pro-healing cytokine expression. Bone formation in a rat calvarial model was accelerated at eight weeks with COL-GL compared to the gold-standard collagen membrane based on microcomputed tomography and histology. Interpenetration of GL13K within collagen sidesteps challenges with antimicrobial coatings on bone regeneration scaffolds while increasing bone regeneration. This strength makes COL-GL a promising approach to reduce post-surgical infections and aid bone regeneration in dental and orthopedic applications. STATEMENT OF SIGNIFICANCE: The COL-GL membrane, incorporating the antimicrobial peptide GL13K within a collagen membrane, signifies a noteworthy breakthrough in bone regeneration strategies for dental and orthopedic applications. By integrating self-assembled GL13K nanofibers into the membrane, this study successfully addresses the challenges associated with antimicrobial coatings, exhibiting improved antimicrobial and anti-fouling activity while preserving compatibility with fibroblasts and pre-osteoblasts. The accelerated bone formation observed in a rat calvarial model emphasizes the potential of this innovative approach to minimize post-surgical infections and enhance bone regeneration outcomes. As a promising alternative for future therapeutic interventions, this material tackles the clinical challenges of extended antibiotic treatments and antibiotic resistance in bone regeneration scenarios.

Chemoselective Coatings of GL13K Antimicrobial Peptides for Dental Implants.

Dental implant-associated infection is a clinical challenge which poses a significant healthcare and socio-economic burden. To overcome this issue, developing antimicrobial surfaces, including antimicrobial peptide coatings, has gained great attention. Different physical and chemical routes have been used to obtain these biofunctional coatings, which in turn might have a direct influence on their bioactivity and functionality. In this study, we present a silane-based, fast, and efficient chemoselective conjugation of antimicrobial peptides (Cys-GL13K) to coat titanium implant surfaces. Comprehensive surface analysis was performed to confirm the surface functionalization of as-prepared and mechanically challenged coatings. The antibacterial potency of the evaluated surfaces was confirmed against both Streptococcus gordonii and Streptococcus mutans, the primary colonizers and pathogens of dental surfaces, as demonstrated by reduced bacteria viability. Additionally, human dental pulp stem cells demonstrated long-term viability when cultured on Cys-GL13K-grafted titanium surfaces. Cell functionality and antimicrobial capability against multi-species need to be studied further; however, our results confirmed that the proposed chemistry for chemoselective peptide anchoring is a valid alternative to traditional site-unspecific anchoring methods and offers opportunities to modify varying biomaterial surfaces to form potent bioactive coatings with multiple functionalities to prevent infection.

Immunomodulatory IL-23 receptor antagonist peptide nanocoatings for implant soft tissue healing.

OBJECTIVE: Peri-implantitis, caused by an inflammatory response to pathogens, is the leading cause of dental implant failure. Poor soft tissue healing surrounding implants - caused by inadequate surface properties - leads to infection, inflammation, and dysregulated keratinocyte and macrophage function. One activated inflammatory response, active around peri-implantitis compared to healthy sites, is the IL-23/IL-17A cytokine axis. Implant surfaces can be synthesized with peptide nanocoatings to present immunomodulatory motifs to target peri-implant keratinocytes to control macrophage polarization and regulate inflammatory axises toward enhancing soft tissue healing. METHODS: We synthesized an IL-23 receptor (IL-23R) noncompetitive antagonist peptide nanocoating using silanization and evaluated keratinocyte secretome changes and macrophage polarization (M1-like "pro-inflammatory" vs. M2-like "pro-regenerative"). RESULTS: IL-23R antagonist peptide nanocoatings were successfully synthesized on titanium, to model dental implant surfaces, and compared to nonfunctional nanocoatings and non-coated titanium. IL-23R antagonist nanocoatings significantly decreased keratinocyte IL-23, and downstream IL-17A, expression compared to controls. This peptide noncompetitive antagonistic function was demonstrated under lipopolysaccharide stimulation. Large scale changes in keratinocyte secretome content, toward a pro-regenerative milieu, were observed from keratinocytes cultured on the IL-23R antagonist nanocoatings compared to controls. Conditioned medium collected from keratinocytes cultured on the IL-23R antagonist nanocoatings polarized macrophages toward a M2-like phenotype, based on increased CD163 and CD206 expression and reduced iNOS expression, compared to controls. SIGNIFICANCE: Our results support development of IL-23R noncompetitive antagonist nanocoatings to reduce the pro-inflammatory IL-23/17A pathway and augment macrophage polarization toward a pro-regenerative phenotype. Immunomodulatory implant surface engineering may promote soft tissue healing and thereby reduce rates of peri-implantitis.

Implant surface physicochemistry affects keratinocyte hemidesmosome formation.

Previous studies have shown hydrophilic/hydrophobic implant surfaces stimulate/hinder osseointegration. An analogous concept was applied here using common biological functional groups on a model surface to promote oral keratinocytes (OKs) proliferation and hemidesmosomes (HD) to extend implant lifespans through increased soft tissue attachment. However, it is unclear what physicochemistry stimulates HDs. Thus, common biological functional groups (NH2 , OH, and CH3 ) were functionalized on glass using silanization. Non-functionalized plasma-cleaned glass and H silanization were controls. Surface modifications were confirmed with X-ray photoelectron spectroscopy and water contact angle. The amount of bovine serum albumin (BSA) and fibrinogen, and BSA thickness, were assessed to understand how adsorbed protein properties were influenced by physicochemistry and may influence HDs. OKs proliferation was measured, and HDs were quantified with immunofluorescence for collagen XVII and integrin ß4. Plasma-cleaned surfaces were the most hydrophilic group overall, while CH3 was the most hydrophobic and OH was the most hydrophilic among functionalized groups. Modification with the OH chemical group showed the highest OKs proliferation and HD expression. The OKs response on OH surfaces appeared to not correlate to the amount or thickness of adsorbed model proteins. These results reveal relevant surface physicochemical features to favor HDs and improve implant soft tissue attachment.