Designing Collagen-Binding Peptide with Enhanced Properties Using Hydropathic Free Energy Predictions.

Collagen is fundamental to a vast diversity of health functions and potential therapeutics. Short peptides targeting collagen are attractive for designing modular systems for site-specific delivery of bioactive agents. Characterization of peptide-protein binding involves a larger number of potential interactions that require screening methods to target physiological conditions. We build a hydropathy-based free energy estimation tool which allows quick evaluation of peptides binding to collagen. Previous studies showed that pH plays a significant role in collagen structure and stability. Our design tool enables probing peptides for their collagen-binding property across multiple pH conditions. We explored binding features of currently known collagen-binding peptides, collagen type I alpha chain 2 sense peptide (TKKTLRT) and decorin LRR-10 (LRELHLNNN). Based on these analyzes, we engineered a collagen-binding peptide with enhanced properties across a large pH range in contrast to LRR-10 pH dependence. To validate our predictions, we used a quantum-dots-based binding assay to compare the coverage of the peptides on type I collagen. The predicted peptide resulted in improved collagen binding. Hydropathy of the peptide-protein pair is a promising approach to finding compatible pairings with minimal use of computational resources, and our method allows for quick evaluation of peptides for binding to other proteins. Overall, the free-energy-based tool provides an alternative computational screening approach that impacts protein interaction search methods.

Engineered Peptides Enable Biomimetic Route for Collagen Intrafibrillar Mineralization.

Overcoming the short lifespan of current dental adhesives remains a significant clinical need. Adhesives rely on formation of the hybrid layer to adhere to dentin and penetrate within collagen fibrils. However, the ability of adhesives to achieve complete enclosure of demineralized collagen fibrils is recognized as currently unattainable. We developed a peptide-based approach enabling collagen intrafibrillar mineralization and tested our hypothesis on a type-I collagen-based platform. Peptide design incorporated collagen-binding and remineralization-mediating properties using the domain structure conservation approach. The structural changes from representative members of different peptide clusters were generated for each functional domain. Common signatures associated with secondary structure features and the related changes in the functional domain were investigated by attenuated total reflectance Fourier-transform infrared (ATR-FTIR) and circular dichroism (CD) spectroscopy, respectively. Assembly and remineralization properties of the peptides on the collagen platforms were studied using atomic force microscopy (AFM). Mechanical properties of the collagen fibrils remineralized by the peptide assemblies was studied using PeakForce-Quantitative Nanomechanics (PF-QNM)-AFM. The engineered peptide was demonstrated to offer a promising route for collagen intrafibrillar remineralization. This approach offers a collagen platform to develop multifunctional strategies that combine different bioactive peptides, polymerizable peptide monomers, and adhesive formulations as steps towards improving the long-term prospects of composite resins.

Spatiotemporal Imaging of Zinc Ions in Zebrafish Live Brain Tissue Enabled by Fluorescent Bionanoprobes.

The zebrafish is a powerful model organism to study the mechanisms governing transition metal ions within whole brain tissue. Zinc is one of the most abundant metal ions in the brain, playing a critical pathophysiological role in neurodegenerative diseases. The homeostasis of free, ionic zinc (Zn2+) is a key intersection point in many of these diseases, including Alzheimer's disease and Parkinson's disease. A Zn2+ imbalance can eventuate several disturbances that may lead to the development of neurodegenerative changes. Therefore, compact, reliable approaches that allow the optical detection of Zn2+ across the whole brain would contribute to our current understanding of the mechanisms that underlie neurological disease pathology. We developed an engineered fluorescence protein-based nanoprobe that can spatially and temporally resolve Zn2+ in living zebrafish brain tissue. The self-assembled engineered fluorescence protein on gold nanoparticles was shown to be confined to defined locations within the brain tissue, enabling site specific studies, compared to fluorescent protein-based molecular tools, which diffuse throughout the brain tissue. Two-photon excitation microscopy confirmed the physical and photometrical stability of these nanoprobes in living zebrafish (Danio rerio) brain tissue, while the addition of Zn2+ quenched the nanoprobe fluorescence. Combining orthogonal sensing methods with our engineered nanoprobes will enable the study of imbalances in homeostatic Zn2+ regulation. The proposed bionanoprobe system offers a versatile platform to couple metal ion specific linkers and contribute to the understanding of neurological diseases.

Autonomous-Strengthening Adhesive Provides Hydrolysis-Resistance and Enhanced Mechanical Properties in Wet Conditions.

The low-viscosity adhesive that is used to bond composite restorative materials to the tooth is readily damaged by acids, enzymes, and oral fluids. Bacteria infiltrate the resulting gaps at the composite/tooth interface, demineralize the tooth, and further erode the adhesive. This paper presents the preparation and characterization of a low-crosslink-density hydrophilic adhesive that capitalizes on sol-gel reactions and free-radical polymerization to resist hydrolysis and provide enhanced mechanical properties in wet environments. Polymerization behavior, water sorption, and leachates were investigated. Dynamic mechanical analyses (DMA) were conducted using water-saturated adhesives to mimic load transfer in wet conditions. Data from all tests were analyzed using appropriate statistical tests (α = 0.05). The degree of conversion was comparable for experimental and control adhesives at 88.3 and 84.3%, respectively. HEMA leachate was significantly lower for the experimental (2.9 wt%) compared to control (7.2 wt%). After 3 days of aqueous aging, the storage and rubbery moduli and the glass transition temperature of the experimental adhesive (57.5MPa, 12.8MPa, and 38.7 °C, respectively) were significantly higher than control (7.4MPa, 4.3 MPa, and 25.9 °C, respectively). The results indicated that the autonomic sol-gel reaction continues in the wet environment, leading to intrinsic reinforcement of the polymer network, improved hydrolytic stability, and enhanced mechanical properties.

Peptide-Enabled Nanocomposites Offer Biomimetic Reconstruction of Silver Diamine Fluoride-Treated Dental Tissues.

Caries is the most ubiquitous infectious disease of mankind, and early childhood caries (ECC) is the most prevalent chronic disease in children worldwide, with the resulting destruction of the teeth recognized as a global health crisis. Recent the United States Food and Drug Administration (FDA) approval for the use of silver diamine fluoride (SDF) in dentistry offers a safe, accessible, and inexpensive approach to arrest caries progression in children with ECC. However, discoloration, i.e., black staining, of demineralized or cavitated surfaces treated with SDF has limited its widespread use. Targeting SDF-treated tooth surfaces, we developed a biohybrid calcium phosphate nanocomposite interface building upon the self-assembly of synthetic biomimetic peptides. Here, an engineered bifunctional peptide composed of a silver binding peptide (AgBP) is covalently joined to an amelogenin derived peptide (ADP). The AgBP provides anchoring to the SDF-treated tooth tissue, while the ADP promotes rapid formation of a calcium phosphate isomorph nanocomposite mimicking the biomineralization function of the amelogenin protein. Our results demonstrate that the bifunctional peptide was effective in remineralizing the biomineral destroyed by caries on the SDF-treated tooth tissues. The proposed engineered peptide approach offers a biomimetic path for remineralization of the SDF-treated tissues producing a calcium phosphate nanocomposite interface competent to be restored using commonly available adhesive dental composites.

Integration of clinical perspective into biomimetic bioreactor design for orthopedics.

The challenges to accommodate multiple tissue formation metrics in conventional bioreactors have resulted in an increased interest to explore novel bioreactor designs. Bioreactors allow researchers to isolate variables in controlled environments to quantify cell response. While current bioreactor designs can effectively provide either mechanical, electrical, or chemical stimuli to the controlled environment, these systems lack the ability to combine all these stimuli simultaneously to better recapitulate the physiological environment. Introducing a dynamic and systematic combination of biomimetic stimuli bioreactor systems could tremendously enhance its clinical relevance in research. Thus, cues from different tissue responses should be studied collectively and included in the design of a biomimetic bioreactor platform. This review begins by providing a summary on the progression of bioreactors from simple to complex designs, focusing on the major advances in bioreactor technology and the approaches employed to better simulate in vivo conditions. The current state of bioreactors in terms of their clinical relevance is also analyzed. Finally, this review provides a comprehensive overview of individual biophysical stimuli and their role in establishing a biomimetic microenvironment for tissue engineering. To date, the most advanced bioreactor designs only incorporate one or two stimuli. Thus, the cell response measured is likely unrelated to the actual clinical performance. Integrating clinically relevant stimuli in bioreactor designs to study cell response can further advance the understanding of physical phenomenon naturally occurring in the body. In the future, the clinically informed biomimetic bioreactor could yield more efficiently translatable results for improved patient care.

Engineering peptide-polymer hybrids for targeted repair and protection of cervical lesions.

By 2060, nearly 100 million people in the U.S. will be over age 65 years. One-third of these older adults will have root caries, and nearly 80% will have dental erosion. These conditions can cause pain and loss of tooth structure that interfere with eating, speaking, sleeping, and quality of life. Current treatments for root caries and dental erosion have produced unreliable results. For example, the glass-ionomer-cement or composite-resin restorations used to treat these lesions have annual failure rates of 44% and 17%, respectively. These limitations and the pressing need to treat these conditions in the aging population are driving a focus on microinvasive strategies, such as sealants and varnishes. Sealants can inhibit caries on coronal surfaces, but they are ineffective for root caries. For healthy, functionally independent elders, chlorhexidine varnish applied every 3 months inhibits root caries, but this bitter-tasting varnish stains the teeth. Fluoride gel inhibits root caries, but requires prescriptions and daily use, which may not be feasible for some older patients. Silver diamine fluoride can both arrest and inhibit root caries but stains the treated tooth surface black. The limitations of current approaches and high prevalence of root caries and dental erosion in the aging population create an urgent need for microinvasive therapies that can: (a) remineralize damaged dentin; (b) inhibit bacterial activity; and (c) provide durable protection for the root surface. Since cavitated and non-cavitated root lesions are difficult to distinguish, optimal approaches will treat both. This review will explore the multi-factorial elements that contribute to root surface lesions and discuss a multi-pronged strategy to both repair and protect root surfaces. The strategy integrates engineered peptides, novel polymer chemistry, multi-scale structure/property characterization and predictive modeling to develop a durable, microinvasive treatment for root surface lesions.

Solid-Binding Peptide-Guided Spatially Directed Immobilization of Kinetically Matched Enzyme Cascades in Membrane Nanoreactors.

Biocatalysis is a useful strategy for sustainable green synthesis of fine chemicals due to its high catalytic rate, reaction specificity, and operation under ambient conditions. Addressable immobilization of enzymes onto solid supports for one-pot multistep biocatalysis, however, remains a major challenge. In natural pathways, enzymes are spatially coupled to prevent side reactions, eradicate inhibitory products, and channel metabolites sequentially from one enzyme to another. Construction of a modular immobilization platform enabling spatially directed assembly of multiple biocatalysts would, therefore, not only allow the development of high-efficiency bioreactors but also provide novel synthetic routes for chemical synthesis. In this study, we developed a modular cascade flow reactor using a generalizable solid-binding peptide-directed immobilization strategy that allows selective immobilization of fusion enzymes on anodic aluminum oxide (AAO) monoliths with high positional precision. Here, the lactate dehydrogenase and formate dehydrogenase enzymes were fused with substrate-specific peptides to facilitate their self-immobilization through the membrane channels in cascade geometry. Using this cascade model, two-step biocatalytic production of l-lactate is demonstrated with concomitant regeneration of soluble nicotinamide adenine dinucleotide (NADH). Both fusion enzymes retained their catalytic activity upon immobilization, suggesting their optimal display on the support surface. The 85% cascading reaction efficiency was achieved at a flow rate that kinetically matches the residence time of the slowest enzyme. In addition, 84% of initial catalytic activity was preserved after 10 days of continuous operation at room temperature. The peptide-directed modular approach described herein is a highly effective strategy to control surface orientation, spatial localization, and loading of multiple enzymes on solid supports. The implications of this work provide insight for the single-step construction of high-power cascadic devices by enabling co-expression, purification, and immobilization of a variety of engineered fusion enzymes on patterned surfaces.

Reconfigurable Dual Peptide Tethered Polymer System Offers a Synergistic Solution for Next Generation Dental Adhesives.

Resin-based composite materials have been widely used in restorative dental materials due to their aesthetic, mechanical, and physical properties. However, they still encounter clinical shortcomings mainly due to recurrent decay that develops at the composite-tooth interface. The low-viscosity adhesive that bonds the composite to the tooth is intended to seal this interface, but the adhesive seal is inherently defective and readily damaged by acids, enzymes, and oral fluids. Bacteria infiltrate the resulting gaps at the composite-tooth interface and bacterial by-products demineralize the tooth and erode the adhesive. These activities lead to wider and deeper gaps that provide an ideal environment for bacteria to proliferate. This complex degradation process mediated by several biological and environmental factors damages the tooth, destroys the adhesive seal, and ultimately, leads to failure of the composite restoration. This paper describes a co-tethered dual peptide-polymer system to address composite-tooth interface vulnerability. The adhesive system incorporates an antimicrobial peptide to inhibit bacterial attack and a hydroxyapatite-binding peptide to promote remineralization of damaged tooth structure. A designer spacer sequence was incorporated into each peptide sequence to not only provide a conjugation site for methacrylate (MA) monomer but also to retain active peptide conformations and enhance the display of the peptides in the material. The resulting MA-antimicrobial peptides and MA-remineralization peptides were copolymerized into dental adhesives formulations. The results on the adhesive system composed of co-tethered peptides demonstrated both strong metabolic inhibition of S. mutans and localized calcium phosphate remineralization. Overall, the result offers a reconfigurable and tunable peptide-polymer hybrid system as next-generation adhesives to address composite-tooth interface vulnerability.

Chemometrics-Assisted Raman Spectroscopy Characterization of Tunable Polymer-Peptide Hybrids for Dental Tissue Repair.

The interfaces that biological tissues form with biomaterials are invariably defective and frequently the location where failure initiates. Characterizing the phenomena that lead to failure is confounded by several factors including heterogeneous material/tissue interfaces. To seamlessly analyze across these diverse structures presents a wealth of analytical challenges. This study aims to develop a molecular-level understanding of a peptide-functionalized adhesive/collagen hybrid biomaterial using Raman spectroscopy combined with chemometrics approach. An engineered hydroxyapatite-binding peptide (HABP) was copolymerized in dentin adhesive and dentin was demineralized to provide collagen matrices that were partially infiltrated with the peptide-functionalized adhesive. Partial infiltration led to pockets of exposed collagen-a condition that simulates defects in adhesive/dentin interfaces. The spectroscopic results indicate that co-polymerizable HABP tethered to the adhesive promoted remineralization of the defects. The spatial distribution of collagen, adhesive, and mineral as well as crystallinity of the mineral across this heterogeneous material/tissue interface was determined using micro-Raman spectroscopy combined with chemometrics approach. The success of this combined approach in the characterization of material/tissue interfaces stems from its ability to extract quality parameters that are related to the essential and relevant portions of the spectral data, after filtering out noise and non-relevant information. This ability is critical when it is not possible to separate components for analysis such as investigations focused on, in situ chemical characterization of interfaces. Extracting essential information from complex bio/material interfaces using data driven approaches will improve our understanding of heterogeneous material/tissue interfaces. This understanding will allow us to identify key parameters within the interfacial micro-environment that should be harnessed to develop durable biomaterials.

Probing Selective Self-Assembly of Putrescine Oxidase with Controlled Orientation Using a Genetically Engineered Peptide Tag.

Controlling enzyme orientation and location on surfaces is a critical step for their successful deployment in diverse applications from biosensors to lab-on-a-chip devices. Functional activity of the enzymes on the surface will largely depend on the spatial arrangement and orientation. Solid binding peptides have been proven to offer versatility for immobilization of biomolecules on inorganic materials including metals, oxides, and minerals. Previously, we demonstrated the utility of a gold binding peptide genetically incorporated into the enzyme putrescine oxidase (PutOx-AuBP), enabling self-enzyme assembly on gold substrates. PutOx is an attractive biocatalyst among flavin oxidases, using molecular oxygen as an electron acceptor without requiring a dissociable coenzyme. Here, we explore the selective self-assembly of this enzyme on a range of surfaces using atomic force microscopy (AFM) along with the assessment of functional activity. This work probes the differences in surface coverage, distribution, size, shape, and activity of PutOx-AuBP in comparison to those of native putrescine oxidase (PutOx) on multiple surfaces to provide insight for material-selective enzymatic assembly. Surfaces investigated include metal (templated-stripped gold (TSG)), oxide (native SiO2 on Si(111)), minerals (mica and graphite), and self-assembled monolayers (SAMs) with a range of hydrophobicity and charge. Supported by both the coverage and the dimensions of immobilized enzymes, our results indicate that of the surfaces investigated, material-selective binding takes place with orientation control only for PutOx-AuBP onto the TSG substrate. These differences are consistent with the measurements of surface-bound enzymatic activities. Substrate-dependent differences observed indicate significant variations in enzyme-surface interactions ranging from peptide-directed self-assembly to enzyme aggregation. The implications of this study provide insight for the fabrication of enzymatic patterns directed by self-assembling peptide tags onto localized surface regions. Enabling functional enzyme-based nanoscale materials offers a fascinating path for utilization of sustainable biocatalysts integrated into multiscale devices.

Combining genetic algorithm with machine learning strategies for designing potent antimicrobial peptides.

BACKGROUND: Current methods in machine learning provide approaches for solving challenging, multiple constraint design problems. While deep learning and related neural networking methods have state-of-the-art performance, their vulnerability in decision making processes leading to irrational outcomes is a major concern for their implementation. With the rising antibiotic resistance, antimicrobial peptides (AMPs) have increasingly gained attention as novel therapeutic agents. This challenging design problem requires peptides which meet the multiple constraints of limiting drug-resistance in bacteria, preventing secondary infections from imbalanced microbial flora, and avoiding immune system suppression. AMPs offer a promising, bioinspired design space to targeting antimicrobial activity, but their versatility also requires the curated selection from a combinatorial sequence space. This space is too large for brute-force methods or currently known rational design approaches outside of machine learning. While there has been progress in using the design space to more effectively target AMP activity, a widely applicable approach has been elusive. The lack of transparency in machine learning has limited the advancement of scientific knowledge of how AMPs are related among each other, and the lack of general applicability for fully rational approaches has limited a broader understanding of the design space. METHODS: Here we combined an evolutionary method with rough set theory, a transparent machine learning approach, for designing antimicrobial peptides (AMPs). Our method achieves the customization of AMPs using supervised learning boundaries. Our system employs in vitro bacterial assays to measure fitness, codon-representation of peptides to gain flexibility of sequence selection in DNA-space with a genetic algorithm and machine learning to further accelerate the process. RESULTS: We use supervised machine learning and a genetic algorithm to find a peptide active against S. epidermidis, a common bacterial strain for implant infections, with an improved aggregation propensity average for an improved ease of synthesis. CONCLUSIONS: Our results demonstrate that AMP design can be customized to maintain activity and simplify production. To our knowledge, this is the first time when codon-based genetic algorithms combined with rough set theory methods is used for computational search on peptide sequences.

Probing the mineralized tissue-adhesive interface for tensile nature and bond strength.

The mechanical performance of the dentin-adhesive interface contributes significantly to the failure of dental composite restorations. Rational material design can lead to enhanced mechanical performance, but this requires accurate characterization of the mechanical behavior at the dentin-adhesive interface. The mechanical performance of the interface is typically characterized using bond strength tests, such as the micro-tensile test. These tests are plagued by multiple limitations including large variations in the test results. The challenges associated with conventional tensile tests limit our ability to unravel the complex relationships that affect mechanical behavior at the dentin-adhesive interface. This study used the diametral compression test to overcome the challenges inherent in conventional bond strength tests. The bovine femur cortical bone tissue was considered as a surrogate material (the mineralized tissue) for human dentin. Two different adhesive formulations, which differed by means of their self-strengthening properties, were studied. The tensile behavior of the mineralized tissue, the adhesive polymer, and the bond strength of the mineralized tissue - adhesive interface was determined using the diametral compression test. The diametral compression test improved the repeatability for both the tensile and bond strength tests. The rate dependent mechanical behavior was observed for both single material and interfacial material systems. The tensile strength and bond strength of the mineralized tissue-adhesive interface was greater for the self-strengthening formulation as compared to the control.

Bioinspired multifunctional adhesive system for next generation bio-additively designed dental restorations.

Resin-based composite has overtaken dental amalgam as the most popular material for the repair of lost or damaged tooth structure. In spite of the popularity, the average composite lifetime is about half that of amalgam restorations. The leading cause of composite-restoration failure is decay at the margin where the adhesive is applied. The adhesive is intended to seal the composite/tooth interface, but the adhesive seal to dentin is fragile and readily degraded by acids, enzymes and other oral fluids. The inherent weakness of this material system is attributable to several factors including the lack of antimicrobial properties, remineralization capabilities and durable mechanical performance - elements that are central to the integrity of the adhesive/dentin (a/d) interfacial seal. Our approach to this problem offers a transition from a hybrid to a biohybrid structure. Discrete peptides are tethered to polymers to provide multi-bio-functional adhesive formulations that simultaneously achieve antimicrobial and remineralization properties. The bio-additive materials design combines several functional properties with the goal of providing an adhesive that will serve as a durable barrier to recurrent decay at the composite/tooth interface. This article provides an overview of our multi-faceted approach which uses peptides tethered to polymers and new polymer chemistries to achieve the next generation adhesive system - an adhesive that provides antimicrobial properties, repair of defective dentin and enhanced mechanical performance.

Evolution of Network Structure and Mechanical Properties in Autonomous-Strengthening Dental Adhesive.

The inherent degradation property of most dental resins in the mouth leads to the long-term release of degradation by-products at the adhesive/tooth interface. The by-products increase the virulence of cariogenic bacteria, provoking a degradative positive-feedback loop that leads to physicochemical and mechanical failure. Photoinduced free-radical polymerization and sol‒gel reactions have been coupled to produce a novel autonomous-strengthening adhesive with enhanced hydrolytic stability. This paper investigates the effect of network structure on time-dependent mechanical properties in adhesives with and without autonomous strengthening. Stress relaxation was conducted under 0.2% strain for 8 h followed by 40 h recovery in water. The stress‒time relationship is analyzed by nonlinear least-squares data-fitting. The fitted Prony series predicts the sample's history under monotonic loading. Results showed that the control failed after the first loading‒unloading‒recovery cycle with permanent deformation. While for the experimental sample, the displacement was almost completely recovered and the Young's modulus increased significantly after the first test cycle. The experimental polymer exhibited higher degree of conversion, lower leachate, and time-dependent stiffening characteristics. The autonomous-strengthening reaction persists in the aqueous environment leading to a network with enhanced resistance to deformation. The results illustrate a rational approach for tuning the viscoelasticity of durable dental adhesives.

Self-Immobilized Putrescine Oxidase Biocatalyst System Engineered with a Metal Binding Peptide.

Flavin oxidases are valuable biocatalysts for the oxidative synthesis of a wide range of compounds, while at the same time reduce oxygen to hydrogen peroxide. Compared to other redox enzymes, their ability to use molecular oxygen as an electron acceptor offers a relatively simple system that does not require a dissociable coenzyme. As such, they are attractive targets for adaptation as cost-effective biosensor elements. Their functional immobilization on surfaces offers unique opportunities to expand their utilization for a wide range of applications. Genetically engineered peptides have been demonstrated as enablers of the functional assembly of biomolecules at solid material interfaces. Once identified as having a high affinity for the material of interest, these peptides can provide a single step bioassembly process with orientation control, a critical parameter for functional immobilization of the enzymes. In this study, for the first time, we explored the bioassembly of a putrescine oxidase enzyme using a gold binding peptide tag. The enzyme was genetically engineered to incorporate a gold binding peptide with an expectation of an effective display of the peptide tag to interact with the gold surface. In this work, the functional activity and expression were investigated, along with the selectivity of the binding of the peptide-tagged enzyme. The fusion enzyme was characterized using multiple techniques, including protein electrophoresis, enzyme activity, and microscopy and spectroscopic methods, to verify the functional expression of the tagged protein with near-native activity. Binding studies using quartz crystal microbalance (QCM), nanoparticle binding studies, and atomic force microscopy studies were used to address the selectivity of the binding through the peptide tag. Surface binding AFM studies show that the binding was selective for gold. Quartz crystal microbalance studies show a strong increase in the affinity of the peptide-tagged protein over the native enzyme, while activity assays of protein bound to nanoparticles provide evidence that the enzyme retained catalytic activity when immobilized. In addition to showing selectivity, AFM images show significant differences in the height of the molecules when immobilized through the peptide tag compared to immobilization of the native enzyme, indicating differences in orientation of the bound enzyme when attached via the affinity tag. Controlling the orientation of surface-immobilized enzymes would further improve their enzymatic activity and impact diverse applications, including oxidative biocatalysis, biosensors, biochips, and biofuel production.

Harnessing biomolecules for bioinspired dental biomaterials.

Dental clinicians have relied for centuries on traditional dental materials (polymers, ceramics, metals, and composites) to restore oral health and function to patients. Clinical outcomes for many crucial dental therapies remain poor despite many decades of intense research on these materials. Recent attention has been paid to biomolecules as a chassis for engineered preventive, restorative, and regenerative approaches in dentistry. Indeed, biomolecules represent a uniquely versatile and precise tool to enable the design and development of bioinspired multifunctional dental materials to spur advancements in dentistry. In this review, we survey the range of biomolecules that have been used across dental biomaterials. Our particular focus is on the key biological activity imparted by each biomolecule toward prevention of dental and oral diseases as well as restoration of oral health. Additional emphasis is placed on the structure-function relationships between biomolecules and their biological activity, the unique challenges of each clinical condition, limitations of conventional therapies, and the advantages of each class of biomolecule for said challenge. Biomaterials for bone regeneration are not reviewed as numerous existing reviews on the topic have been recently published. We conclude our narrative review with an outlook on the future of biomolecules in dental biomaterials and potential avenues of innovation for biomaterial-based patient oral care.

Mitigation of peri-implantitis by rational design of bifunctional peptides with antimicrobial properties.

The integration of molecular and cell biology with materials science has led to strategies to improve the interface between dental implants with the surrounding soft and hard tissues in order to replace missing teeth and restore mastication. More than 3 million implants have been placed in the US alone and this number is rising by 500,000/year. Peri-implantitis, an inflammatory response to oral pathogens growing on the implant surface threatens to reduce service life leading to eventual implant failure, and such an outcome will have adverse impact on public health and create significant health care costs. Here we report a predictive approach to peptide design, which enabled us to engineer a bifunctional peptide to combat bacterial colonization and biofilm formation, reducing the adverse host inflammatory immune response that destroys the tissue surrounding implants and shortens their lifespans. This bifunctional peptide contains a titanium-binding domain that recognizes and binds with high affinity to titanium implant surfaces, fused through a rigid spacer domain with an antimicrobial domain. By varying the antimicrobial peptide domain, we were able to predict the properties of the resulting bifunctional peptides in their entirety by analyzing the sequence-structure-function relationship. These bifunctional peptides achieve: 1) nearly 100% surface coverage within minutes, a timeframe suitable for their clinical application to existing implants; 2) nearly 100% binding to a titanium surface even in the presence of contaminating serum protein; 3) durability to brushing with a commercially available electric toothbrush; and 4) retention of antimicrobial activity on the implant surface following bacterial challenge. A bifunctional peptide film can be applied to both new implants and/or repeatedly applied to previously placed implants to control bacterial colonization mitigating peri-implant disease that threatens dental implant longevity.

Antimicrobial Peptide-Polymer Conjugates for Dentistry.

Bacterial adhesion and growth at the composite/adhesive/tooth interface remain the primary cause of dental composite restoration failure. Early colonizers, including Streptococcus mutans, play a critical role in the formation of dental caries by creating an environment that reduces the adhesive's integrity. Subsequently, other bacterial species, biofilm formation, and lactic acid from S. mutans demineralize the adjoining tooth. Because of their broad spectrum of antibacterial activity and low risk for antibiotic resistance, antimicrobial peptides (AMPs) have received significant attention to prevent bacterial biofilms. Harnessing the potential of AMPs is still very limited in dentistry-a few studies have explored peptide-enabled antimicrobial adhesive copolymer systems using mainly nonspecific adsorption. In the current investigation, to avoid limitations from nonspecific adsorption and to prevent potential peptide leakage out of the resin, we conjugated an AMP with a commonly used monomer for dental adhesive formulation. To tailor the flexibility between the peptide and the resin material, we designed two different spacer domains. The spacer-integrated antimicrobial peptides were conjugated to methacrylate (MA), and the resulting MA-AMP monomers were next copolymerized into dental adhesives as AMP-polymer conjugates. The resulting bioactivity of the polymethacrylate-based AMP conjugated matrix activity was investigated. The antimicrobial peptide conjugated to the resin matrix demonstrated significant antimicrobial activity against S. mutans. Secondary structure analyses of conjugated peptides were applied to understand the activity differential. When mechanical properties of the adhesive system were investigated with respect to AMP and cross-linking concentration, resulting AMP-polymer conjugates maintained higher compressive moduli compared to hydrogel analogues including polyHEMA. Overall, our result provides a robust approach to develop a fine-tuned bioenabled peptide adhesive system with improved mechanical properties and antimicrobial activity. The results of this study represent a critical step toward the development of peptide-conjugated dentin adhesives for treatment of secondary caries and the enhanced durability of dental composite restorations.