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.

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.

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.

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.

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.

Human Whole Blood Interactions with Craniomaxillofacial Reconstruction Materials: Exploring In Vitro the Role of Blood Cascades and Leukocytes in Early Healing Events.

The present study investigated early interactions between three alloplastic materials (calcium phosphate (CaP), titanium alloy (Ti), and polyetheretherketone (PEEK) with human whole blood using an established in vitro slide chamber model. After 60 min of contact with blood, coagulation (thrombin-antithrombin complexes, TAT) was initiated on all test materials (Ti > PEEK > CaP), with a significant increase only for Ti. All materials showed increased contact activation, with the KK-AT complex significantly increasing for CaP (p < 0.001), Ti (p < 0.01), and PEEK (p < 0.01) while only CaP demonstrated a notable rise in KK-C1INH production (p < 0.01). The complement system had significant activation across all materials, with CaP (p < 0.0001, p < 0.0001) generating the most pronounced levels of C3a and sC5b-9, followed by Ti (p < 0.001, p < 0.001) and lastly, PEEK (p < 0.001, p < 0.01). This activation correlated with leukocyte stimulation, particularly myeloperoxidase release. Consequently, the complement system may assume a more significant role in the early stages post implantation in response to CaP materials than previously recognized. Activation of the complement system and the inevitable activation of leukocytes might provide a more favorable environment for tissue remodeling and repair than has been traditionally acknowledged. While these findings are limited to the early blood response, complement and leukocyte activation suggest improved healing outcomes, which may impact long-term clinical outcomes.

Keratinocyte Proliferation and Hemidesmosome Formation on Surfaces for Dental Implants: In Vitro Study.

PURPOSE: To demonstrate the likelihood of the polyetheretherketone (PEEK), zirconia (ZrO2), and titanium (Ti) disks to support proliferation and hemidesmosome formation of gingival cells. MATERIALS AND METHODS: Water contact angle was performed on each material, and surface roughness (Ra) was measured. Scanning electron microscopy and x-ray photoelectron spectroscopy were used. Later, oral keratinocyte cells were cultured on disks, and metabolic activity and expression of hemidesmosome markers, integrin α6 and ß4, in relation to the biomaterial disks at 1, 3, and 5 days of cell culture were quantified. Tissue culture polystyrene was used as the control. Statistical analysis was performed with analysis of variance (ANOVA) with Tukey post hoc comparison test. A P value of < .05 was considered statistically significant. RESULTS: The water contact angle ranged from 70.2 degrees (Ti) to a maximum of hydrophobicity of 93.3 degrees (PEEK). Ra was highest on ZrO2, followed by PEEK. Ti showed the most keratinocyte metabolic activity at 1, 3, and 5 culture periods. Contrarily, ZrO2 and PEEK disks had lower keratinocyte metabolic activity at all observation times, with no statistical differences between both groups. Integrin α6 and ß4 expression was highest on TCPS and ZrO2 compared to Ti and PEEK. CONCLUSION: Keratinocytes proliferated faster on Ti than on ZrO2 and PEEK substrates, and expression of hemidesmosome formation markers, integrin α6 and ß4, were higher on ZrO2 than either Ti or PEEK. Int J Oral Maxillofac Implants 2023;38:496-502. doi: 10.11607/jomi.9894.

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.

Biomimetic Mineralization of Fibrillar Collagen with Strontium-doped Hydroxyapatite.

Fibrillar collagen structures mineralized with hydroxyapatite using the polymer-induced liquid precursor (PILP) process have been explored as synthetic models for studying biomineralization of human hard tissues and have also been applied in the fabrication of scaffolds for hard tissue regeneration. Strontium has important biological functions in bone and has been used as a therapeutic agent for treating diseases that result in bone defects, such as osteoporosis. Here, we developed a strategy to mineralize collagen with Sr-doped hydroxyapatite (HA) using the PILP process. Doping with Sr altered the crystal lattice of HA and inhibited the degree of mineralization in a concentration-dependent manner, but did not affect the unique formation of intrafibrillar minerals using the PILP. The Sr-doped HA nanocrystals were aligned in the [001] direction but did not recapitulate the parallel alignment of the c-axis of pure Ca HA in relation to the collagen fiber long axis. The mimicry of doping Sr in PILP-mineralized collagen can help understand the doping of Sr in natural hard tissues and during treatment. The fibrillary mineralized collagen with Sr-doped HA will be explored in future work as biomimetic and bioactive scaffolds for regeneration of bone and tooth dentin.

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.

Functional signatures of ex-vivo dental caries onset.

Background: The etiology of dental caries remains poorly understood. With the advent of next-generation sequencing, a number of studies have focused on the microbial ecology of the disease. However, taxonomic associations with caries have not been consistent. Researchers have also pursued function-centric studies of the caries microbial communities aiming to identify consistently conserved functional pathways. A major question is whether changes in microbiome are a cause or a consequence of the disease. Thus, there is a critical need to define conserved functional signatures at the onset of dental caries. Methods: Since it is unethical to induce carious lesions clinically, we developed an innovative longitudinal ex-vivo model integrated with the advanced non-invasive multiphoton second harmonic generation bioimaging to spot the very early signs of dental caries, combined with 16S rRNA short amplicon sequencing and liquid chromatography-mass spectrometry-based targeted metabolomics. Findings: For the first time, we induced longitudinally monitored caries lesions validated with the scanning electron microscope. Consequently, we spotted the caries onset and, associated with it, distinguished five differentiating metabolites - Lactate, Pyruvate, Dihydroxyacetone phosphate, Glyceraldehyde 3-phosphate (upregulated) and Fumarate (downregulated). Those metabolites co-occurred with certain bacterial taxa; Streptococcus, Veillonella, Actinomyces, Porphyromonas, Fusobacterium, and Granulicatella, regardless of the abundance of other taxa. Interpretation: These findings are crucial for understanding the etiology and dynamics of dental caries, and devising targeted interventions to prevent disease progression.

Strontium- and peptide-modified silicate nanostructures for dual osteogenic and antimicrobial activity.

Developing multifunctional nanostructures that promote bone repair while fighting infection is highly desirable in bone regenerative therapies. Previous efforts have focused on achieving one property or another by altering the chemical makeup of nanostructures or using growth factors or antibiotics. We present nanostructures with several simultaneous functional attributes including positive effects of strontium on bone formation and prevention of osteoclast differentiation along with incorporation of antimicrobial peptides (AMP) to prevent infection. To form these multifunctional nanostructures, mesoporous calcium silicate (CaMSN) was modified with high levels of strontium. For this, CaMSNs were either partially substituted (20 wt% Ca) or completely replaced with strontium (Sr) to form Sr-CaMSN or SrMSN. The mesoporous nature of these bioactive silicate nanostructures rendered a configuration for substantial AMP loading as well as their effective delivery. The physico-chemical and structural characterization of synthesized MSNs confirmed the mesoporous nature of the synthesized MSNs and their total surface area, pore size, pore volume and SBF-mediated bioactivity remained unaltered with the incorporation of Sr. However, biological evaluation confirmed that synthesized SrMSN upregulated osteogenic differentiation of mesenchymal stromal cells and significantly downregulated osteoclast differentiation. Also, the AMP-loaded MSNs prevented formation and growth of methicillin resistant Staphylococcus aureus (MRSA) biofilms. Thus, high Sr-containing AMP-loaded SrMSNs may combat MRSA-associated infection while promoting bone regeneration. The controlled availability of therapeutic Sr and AMP release as SrMSN degrade enables its potential application in bone tissue regeneration.

Gelatin Methacryloyl Hydrogels for Musculoskeletal Tissue Regeneration.

Musculoskeletal disorders are a significant burden on the global economy and public health. Hydrogels have significant potential for enhancing the repair of damaged and injured musculoskeletal tissues as cell or drug delivery systems. Hydrogels have unique physicochemical properties which make them promising platforms for controlling cell functions. Gelatin methacryloyl (GelMA) hydrogel in particular has been extensively investigated as a promising biomaterial due to its tuneable and beneficial properties and has been widely used in different biomedical applications. In this review, a detailed overview of GelMA synthesis, hydrogel design and applications in regenerative medicine is provided. After summarising recent progress in hydrogels more broadly, we highlight recent advances of GelMA hydrogels in the emerging fields of musculoskeletal drug delivery, involving therapeutic drugs (e.g., growth factors, antimicrobial molecules, immunomodulatory drugs and cells), delivery approaches (e.g., single-, dual-release system), and material design (e.g., addition of organic or inorganic materials, 3D printing). The review concludes with future perspectives and associated challenges for developing local drug delivery for musculoskeletal applications.

Junctional epithelium and hemidesmosomes: Tape and rivets for solving the "percutaneous device dilemma" in dental and other permanent implants.

The percutaneous device dilemma describes etiological factors, centered around the disrupted epithelial tissue surrounding non-remodelable devices, that contribute to rampant percutaneous device infection. Natural percutaneous organs, in particular their extracellular matrix mediating the "device"/epithelium interface, serve as exquisite examples to inspire longer lasting long-term percutaneous device design. For example, the tooth's imperviousness to infection is mediated by the epithelium directly surrounding it, the junctional epithelium (JE). The hallmark feature of JE is formation of hemidesmosomes, cell/matrix adhesive structures that attach surrounding oral gingiva to the tooth's enamel through a basement membrane. Here, the authors survey the multifaceted functions of the JE, emphasizing the role of the matrix, with a particular focus on hemidesmosomes and their five main components. The authors highlight the known (and unknown) effects dental implant - as a model percutaneous device - placement has on JE regeneration and synthesize this information for application to other percutaneous devices. The authors conclude with a summary of bioengineering strategies aimed at solving the percutaneous device dilemma and invigorating greater collaboration between clinicians, bioengineers, and matrix biologists.

Guiding bone formation using semi-onlay calcium phosphate implants in an ovine calvarial model.

The restoration of cranio-maxillofacial deformities often requires complex reconstructive surgery in a challenging anatomical region, with abnormal soft tissue structures and bony deficits. In this proof-of-concept, the possibility of vertical bone augmentation was explored by suspending hemispherically shaped titanium-reinforced porous calcium phosphate (CaP) implants (n = 12) over the frontal bone in a sheep model (n = 6). The animals were euthanized after week 13 and the specimens were subject to micro-computed tomography (µCT) and comprehensive histological analysis. Histology showed that the space between implant and the recipient bone was filled with a higher percentage of newly formed bone (NFB) versus soft tissue with a median of 53% and 47%, respectively. Similar results were obtained from the µ-CT analysis, with a median of 56% NFB and 44% soft tissue filling the void. Noteworthy, significantly higher bone-implant contact was found for the CaP (78%, range 14%-94%) versus the Titanium (29%, range 0%-75%) portion of the implant exposed to the surrounding bone. The histological analysis indicates that the CaP replacement by bone is driven by macrophages over time, emphasized by material-filled macrophages found in close vicinity to the CaP with only a small number of single osteoclasts found actively remodeling the NFB. This study shows that CaP based implants can be assembled with the help of additive manufacturing to guide vertical bone formation without decortification or administration of growth factors. Furthermore, it highlights the potential disadvantage of a seamless fit between the implant and the recipient's bone.

Dual keratinocyte-attachment and anti-inflammatory coatings for soft tissue sealing around transmucosal oral implants.

Unlike the attachment of soft epithelial skin tissue to penetrating solid natural structures like fingernails and teeth, sealing around percutaneous/permucosal devices such as dental implants is hindered by inflammation and epidermal down growth. Here, we employed a dual keratinocyte-adhesive peptide and anti-inflammatory biomolecule coating on titanium to promote oral epithelial tissue attachment. For minimizing inflammation-triggered epidermal down growth, we coated pristine and oxygen plasma pre-treated polished titanium (pTi) with conjugated linoleic acid (CLA). Further, in order to aid in soft tissue attachment via the formation of hemidesmosomes, adhesive structures by oral keratinocytes, we coated the anionic linoleic acid (LA) adsorbed titanium with cationic cell adhesive peptides (CAP), LamLG3, a peptide derived from Laminin 332, the major extracellular matrix component of the basement membrane in skin tissue and Net1, derived from Netrin-1, a neural chemoattractant capable of epithelial cell attachment via α6ß4 integrins. The dual CLA-CAP coatings on pTi were characterized by X-ray photoelectron spectroscopy and dynamic water contact angle measurements. The proliferation of human oral keratinocytes (TERT-2/OKF6) was accelerated on the peptide coated titanium while also promoting the expression of Col XVII and ß-4 integrin, two markers for hemidesmosomes. Simultaneously, CLA coating suppressed the production of inducible nitric oxide synthase (anti-iNOS); a pro-inflammatory M1 marker expressed in lipopolysaccharide (LPS) stimulated murine macrophages (RAW 264.7) and elevated expression of anti-CD206, associated to an anti-inflammatory M2 macrophage phenotype. Taken together, the dual keratinocyte-adhesive peptide and anti-inflammatory biomolecule coating on titanium can help reduce inflammation and promote permucosal/peri-implant soft tissue sealing.

Utilizing a degradation prediction pathway system to understand how a novel methacrylate derivative polymer with flipped external ester groups retains physico-mechanical properties following esterase exposure.

OBJECTIVE: The region of failure for current methacrylates (i.e. derivatives of acrylates) are ester bond linkages that hydrolyze in the presence of salivary and bacterial esterases that break the polymer network backbone. This effect decreases the mechanical properties of methacrylate-based materials. METHODS: The ethylene glycol dimethacrylate (EGDMA) or novel ethylene glycol ethyl methacrylate (EGEMA) discs were prepared using 40 µL of the curing mixture containing photo/co-initiators for 40 s in a PTFE mold at 1000 mW/cm2. The degree of conversion was used as a quality control measure for the prepared discs, followed by physical, mechanical, and chemical characterization of discs properties before and after cholesterol esterase treatment. RESULTS: After 9 weeks of standardized cholesterol esterase (CEase) exposure, EGDMA discs showed exponential loss of material (p = 0.0296), strength (p = 0.0014) and increased water sorption (p = 0.0002) compared to EGEMA discs. We integrated a degradation prediction pathway system to LC/MS and GC/MS analyses to elucidate the degradation by-products of both EGEMA and EGDMA polymers. GC/MS analysis demonstrated that the esterase catalysis was directed to central polymer backbone breakage, producing ethylene glycol, for EGDMA, and to side chain breakage, producing ethanol, for EGEMA. The flipped external ester group linkage design is attributed to EGEMA showing higher resistance to esterase biodegradation and changes in mechanical and physical properties than EGDMA. SIGNIFICANCE: EGEMA is a potential substitute for common macromer diluents, such as EGDMA, based on its resistance to biodegradation effects. This work inspires the flipped external group design to be applied to analogs of current larger, hydrophobic strength bearing macromers used in future dental material formulations.

Tapping basement membrane motifs: Oral junctional epithelium for surface-mediated soft tissue attachment to prevent failure of percutaneous devices.

Teeth, long-lasting percutaneous organs, feature soft tissue attachment through adhesive structures, hemidesmosomes, in the junctional epithelium basement membrane adjacent to teeth. This soft tissue attachment prevents bacterial infection of the tooth despite the rich - and harsh - microbial composition of the oral cavity. Conversely, millions of percutaneous devices (catheters, dental, and orthopedic implants) fail from infection yearly. Standard of care antibiotic usage fuels antimicrobial resistance and is frequently ineffective. Infection prevention strategies, like for dental implants, have failed in generating durable soft tissue adhesion - like that seen with the tooth - to prevent bacterial colonization at the tissue-device interface. Here, inspired by the impervious natural attachment of the junctional epithelium to teeth, we synthesized four cell adhesion peptide (CAPs) nanocoatings, derived from basement membranes, to promote percutaneous device soft tissue attachment. The two leading nanocoatings upregulated integrin-mediated hemidesmosomes, selectively increased keratinocyte proliferation compared to fibroblasts, which cannot form hemidesmosomes, and expression of junctional epithelium adhesive markers. CAP nanocoatings displayed marked durability under simulated clinical conditions and the top performer CAP nanocoating was validated in a percutaneous implant murine model. Basement membrane CAP nanocoatings, inspired by the tooth and junctional epithelium, may provide an alternative anti-infective strategy for percutaneous devices to mitigate the worldwide threat of antimicrobial resistance. STATEMENT OF SIGNIFICANCE: Prevention and management of medical device infection is a significant healthcare challenge. Overzealous antibiotic use has motivated alternative material innovations to prevent infection. Here, we report implant cell adhesion peptide nanocoatings that mimic a long-lasting, natural "medical device," the tooth, through formation of cell adhesive structures called hemidesmosomes. Such nanocoatings sidestep the use of antimicrobial or antibiotic elements to form a soft-tissue seal around implants. The top performing nanocoatings prompted expression of hemidesmosomes and defensive factors to mimic the tooth and was validated in an animal model. Application of cell adhesion peptide nanocoatings may provide an alternative to preventing, rather that necessarily treating, medical device infection across a range of device indications, like dental implants.

Hybrid nanocoatings of self-assembled organic-inorganic amphiphiles for prevention of implant infections.

Antimicrobial coatings are one of the most promising strategies to prevent bacterial infections in orthopedic and dental implants. Combining antimicrobial agents with different antimicrobial mechanisms might have synergistic effects and be more potent. Others have shown that nanocomposites of silver nanoparticles (AgNPs) decorated with antimicrobial peptides (AMPs) show increased potency as free agents in solution. However, similar nanocomposites have not been explored to coat biomaterials through cooperative weak electrostatic attraction forces between AMP, AgNPs and substrates in need of protection against infection. In this work, we synthesized self-assembled antimicrobial amphiphiles of an AMP, GL13K. Then, we decorated the AMP nanostructures with AgNPs, which were finally used to coat etched Ti (eTi) surfaces. The strong hydrogen bonding between the AMP amphiphiles and the polar eTi yielded a robust and stable coating. When compared to single AgNP or single AMP coatings, our hybrid nanocoatings had notably higher in vitro antimicrobial potency against multiple bacteria strains related to implant infection. The hybrid coating also showed relevant antimicrobial activity in an in vivo subcutaneous infection model in rats. This work advances the application of AgNP/AMP nanocomposites as effective coatings for prevention of implant infections. STATEMENT OF SIGNIFICANCE: High morbidity, mortality and elevated costs are associated with orthopedic and dental implant infections. Conventional antibiotic treatment is ineffective due to barrier-like extracellular polymeric substances in biofilms and the increasing threat from antibiotic resistance. Antimicrobial coatings are one of the most promising strategies, but the performance is usually unsatisfactory, especially when tested in vivo. Here, we present a hybrid nanocoating with different modes of action to prevent implant infections using self-assembled antimicrobial peptide (AMP) amphiphiles decorated with silver nanoparticles (AgNPs). When compared to single AgNP or AMP coatings, our hybrid nanocoatings showed significant increases in antimicrobial potency against multiple implant infection-related bacterial strains in vitro and in an in vivo rat subcutaneous infection model.