Nitric oxide releasing coatings for the prevention of viral and bacterial infections.

Healthcare associated infections (HCAI) represent a significant burden worldwide contributing to morbidity and mortality and result in substantial economic consequences equating to billions annually. Although the impacts of HCAI have been felt for many years, the coronavirus pandemic has had a profound effect, escalating rates of HCAI, even with extensive preventative measures such as vaccination, personal protective equipment, and deep cleaning regimes. Therefore, there is an urgent need for new solutions to mitigate this serious health emergency. In this paper, the fabrication of nitric oxide (NO) releasing dual action polymer coatings for use in healthcare applications is described. The coatings are doped with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) and release high payloads of NO in a sustained manner for in excess of 50 hours. These coatings are extensively characterized in multiple biologically relevant solutions and the antibacterial/antiviral efficacy is studied. For the first time, we assess antibacterial activity in a time course study (1, 2, 4 and 24 h) in both nutrient rich and nutrient poor conditions. Coatings exhibit excellent activity against Pseudomonas aeruginosa and methicillin resistant Staphylococcus aureus (MRSA), with up to complete reduction observed over 24 hours. Additionally, when tested against SARS-CoV-2, the coatings significantly reduced active virus in as little as 10 minutes. These promising results suggest that these coatings could be a valuable addition to existing preventative measures in the fight against HCAIs.

Cryo-Electrospinning Generates Highly Porous Fiber Scaffolds Which Improves Trabecular Meshwork Cell Infiltration.

Human trabecular meshwork is a sieve-like tissue with large pores, which plays a vital role in aqueous humor outflow. Dysfunction of this tissue can occur, which leads to glaucoma and permanent vision loss. Replacement of trabecular meshwork with a tissue-engineered device is the ultimate objective. This study aimed to create a biomimetic structure of trabecular meshwork using electrospinning. Conventional electrospinning was compared to cryogenic electrospinning, the latter being an adaptation of conventional electrospinning whereby dry ice is incorporated in the fiber collector system. The dry ice causes ice crystals to form in-between the fibers, increasing the inter-fiber spacing, which is retained following sublimation. Structural characterization demonstrated cryo-scaffolds to have closer recapitulation of the trabecular meshwork, in terms of pore size, porosity, and thickness. The attachment of a healthy, human trabecular meshwork cell line (NTM5) to the scaffold was not influenced by the fabrication method. The main objective was to assess cell infiltration. Cryo-scaffolds supported cell penetration deep within their structure after seven days, whereas cells remained on the outer surface for conventional scaffolds. This study demonstrates the suitability of cryogenic electrospinning for the close recapitulation of trabecular meshwork and its potential as a 3D in vitro model and, in time, a tissue-engineered device.

Aminosilane Functionalized Aligned Fiber PCL Scaffolds for Peripheral Nerve Repair.

Silane modification is a simple and cost-effective tool to modify existing biomaterials for tissue engineering applications. Aminosilane layer deposition has previously been shown to control NG108-15 neuronal cell and primary Schwann cell adhesion and differentiation by controlling deposition of ─NH2 groups at the submicron scale across the entirety of a surface by varying silane chain length. This is the first study toreport depositing 11-aminoundecyltriethoxysilane (CL11) onto aligned Polycaprolactone (PCL) scaffolds for peripheral nerve regeneration. Fibers are manufactured via electrospinning and characterized using water contact angle measurements, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). Confirmed modified fibers are investigated using in vitro cell culture of NG108-15 neuronal cells and primary Schwann cells to determine cell viability, cell differentiation, and phenotype. CL11-modified fibers significantly support NG108-15 neuronal cell and Schwann cell viability. NG108-15 neuronal cell differentiation maintains Schwann cell phenotype compared to unmodified PCL fiber scaffolds. 3D ex vivo culture of Dorsal root ganglion explants (DRGs) confirms further Schwann cell migration and longer neurite outgrowth from DRG explants cultured on CL11 fiber scaffolds compared to unmodified scaffolds. Thus, a reproducible and cost-effective tool is reported to modify biomaterials with functional amine groups that can significantly improve nerve guidance devices and enhance nerve regeneration.

Antimicrobial Nitric Oxide-Releasing Electrospun Dressings for Wound Healing Applications.

Nonhealing and chronic wounds represent a major problem for the quality of life of patients and have cost implications for healthcare systems. The pathophysiological mechanisms that prevent wound healing are usually multifactorial and relate to patient overall health and nutrition, vascularity of the wound bed, and coexisting infection/colonization. Bacterial infections are one of the predominant issues that can stall a wound, causing it to become chronic. Successful wound healing often depends on weeks or months of antimicrobial therapy, but this is problematic given the rise in multidrug-resistant bacteria. As such, alternatives to antibiotics are desperately needed to aid the healing of chronic, and even acutely infected wounds. Nitric oxide (NO) kills bacteria through a variety of mechanisms, and thus, bacteria have shown no tendency to develop resistance to NO as a therapeutic agent and therefore can be a good alternative to antibiotic therapy. In this paper, we report on the development of NO-releasing electrospun membranes fabricated from polycaprolactone (PCL)/gelatin blends and optimized to reduce bacterial infection. The NO payload in the membranes was directly related to the number of amines (and hence the amount of gelatin) in the blend. Higher NO payloads corresponded with a higher degree of antimicrobial efficacy. No cytotoxicity was observed for electrospun membranes, and an in vitro wound closure assay demonstrated closure within 16 h. The results presented here clearly indicate that these NO-releasing electrospun membranes hold significant promise as wound dressings due to their antimicrobial activity and biocompatibility.

Melt electro-written scaffolds with box-architecture support orthogonally oriented collagen.

Melt electro-writing (MEW) is a state-of-the-art technique that supports fabrication of 3D, precisely controlled and reproducible fiber structures. A standard MEW scaffold design is a box-structure, where a repeat layer of 90° boxes is produced from a single fiber. In 3D form (i.e. multiple layers), this structure has the potential to mimic orthogonal arrangements of collagen, as observed in the corneal stroma. In this study, we determined the response of human primary corneal stromal cells and their deposited fibrillar collagen (detected using a CNA35 probe) following six weeksin vitroculture on these box-structures made from poly(ϵ-caprolactone) (PCL). Comparison was also made to glass substrates (topography-free) and electrospun PCL fibers (aligned topography). Cell orientation and collagen deposition were non-uniform on glass substrates. Electrospun scaffolds supported an excellent parallel arrangement of cells and deposited collagen to the underlying architecture of aligned fibers, but there was no evidence of bidirectional collagen. In contrast, MEW scaffolds encouraged the formation of a dense, interconnected cellular network and deposited fibrillar collagen layers with a distinct orthogonal-arrangement. Collagen fibrils were particularly dominant through the middle layers of the MEW scaffolds' total thickness and closer examination revealed these fibrils to be concentrated within the pores' central regions. With the demand for donor corneas far exceeding the supply-leaving many with visual impairment-the application of MEW as a potential technique to recreate the corneal stroma with spontaneous, bidirectional collagen organization warrants further study.

Cost effective optimised synthetic surface modification strategies for enhanced control of neuronal cell differentiation and supporting neuronal and Schwann cell viability.

Enriching a biomaterial surface with specific chemical groups has previously been considered for producing surfaces that influence cell response. Silane layer deposition has previously been shown to control mesenchymal stem cell adhesion and differentiation. However, it has not been used to investigate neuronal or Schwann cell responses in vitro to date. We report on the deposition of aminosilane groups for peripheral neurons and Schwann cells studying two chain lengths: (a) 3-aminopropyl triethoxysilane (short chain-SC) and (b) 11-aminoundecyltriethoxysilane (long chain-LC) by coating glass substrates. Surfaces were characterised by water contact angle, AFM and XPS. LC-NH2 was produced reproducibly as a homogenous surface with controlled nanotopography. Primary neuron and NG108-15 neuronal cell differentiation and primary Schwann cell responses were investigated in vitro by S100ß, p75, and GFAP antigen expression. Both amine silane surface supported neuronal and Schwann cell growth; however, neuronal differentiation was greater on LC aminosilanes versus SC. Thus, we report that silane surfaces with an optimal chain length may have potential in peripheral nerve repair for the modification and improvement of nerve guidance devices.

Material Characterisation and Stratification of Conjunctival Epithelial Cells on Electrospun Poly(ε-Caprolactone) Fibres Loaded with Decellularised Tissue Matrices.

The conjunctiva, an under-researched yet incredibly important tissue, plays key roles in providing protection to the eye and maintaining homeostasis of its ocular surface. Multiple diseases can impair conjunctival function leading to severe consequences that require surgical intervention. Small conjunctival defects can be repaired relatively easily, but larger defects rely on tissue grafts which generally do not provide adequate healing. A tissue engineering approach involving a biomaterial substrate capable of supporting a stratified epithelium with embedded, mucin-secreting goblet cells offers a potential solution. As a first step, this study aimed to induce stratification of human conjunctival epithelial cells cultured on electrospun scaffolds composed from poly(ε-caprolactone) (PCL) and decellularised tissue matrix (small intestinal submucosa (SIS) or urinary bladder matrix (UBM)) and held at the air/liquid interface. Stratification, up to 5 cell layers, occurred more frequently on scaffolds containing PCL + UBM. Incorporation of these decellularised tissue matrices also impacted material properties, with significant changes occurring to their fibre diameter, tensile properties, and chemical composition throughout the scaffold structure compared to PCL alone. These matrix containing scaffolds warrant further long-term investigation as a potential advanced therapy medicinal product for conjunctiva repair and regeneration.

Exploiting biomaterial approaches to manufacture an artificial trabecular meshwork: A progress report.

Glaucoma is the second leading cause of irreversible blindness worldwide. Glaucoma is a progressive optic neuropathy in which permanent loss of peripheral vision results from neurodegeneration in the optic nerve head. The trabecular meshwork is responsible for regulating intraocular pressure, which to date, is the only modifiable risk factor associated with the development of glaucoma. Lowering intraocular pressure reduces glaucoma progression and current surgical approaches for glaucoma attempt to reduce outflow resistance through the trabecular meshwork. Many surgical approaches use minimally invasive glaucoma surgeries (MIGS) to control glaucoma. In this progress report, biomaterials currently employed to treat glaucoma, such as MIGS, and the issues associated with them are described. The report also discusses innovative biofabrication approaches that aim to revolutionise glaucoma treatment through tissue engineering and regenerative medicine (TERM). At present, there are very few applications targeted towards TM engineering in vivo, with a great proportion of these biomaterial structures being developed for in vitro model use. This is a consequence of the many anatomical and physiological attributes that must be considered when designing a TERM device for microscopic tissues, such as the trabecular meshwork. Ongoing advancements in TERM research from multi-disciplinary teams should lead to the development of a state-of-the-art device to restore trabecular meshwork function and provide a bio-engineering solution to improve patient outcomes.

Nitric Oxide Releasing Titanium Surfaces for Antimicrobial Bone-Integrating Orthopedic Implants.

Titanium implants in orthopedic applications can fail due to infection and impaired integration into the host. Most research efforts that facilitate osseointegration of the implant have not considered infection, and vice versa. Moreover, most infection control measures involve the use of conventional antibiotics which contributes to the global epidemic of antimicrobial resistance. Nitric oxide (NO) is a promising alternative to antibiotics, and while researchers have investigated NO releasing coatings, there are few reports on the function/robustness or the mechanism of NO release. Our comprehensive mechanistic study has allowed us to design, characterize, and optimize NO releasing coatings to achieve maximum antimicrobial efficacy toward bacteria with minimum cytotoxicity to human primary osteoblasts in vitro. As the antibiotic era is coming to an end and the future of infection control continues to demand new alternatives, the coatings described herein represent a promising therapeutic strategy for use in orthopedic surgeries.

Effect of Polymer Demixed Nanotopographies on Bacterial Adhesion and Biofilm Formation.

As the current global threat of antimicrobial resistance (AMR) persists, developing alternatives to antibiotics that are less susceptible to resistance is becoming an urgent necessity. Recent advances in biomaterials have allowed for the development and fabrication of materials with discrete surface nanotopographies that can deter bacteria from adhering to their surface. Using binary polymer blends of polystyrene (PS), poly(methyl methacrylate) (PMMA) and polycaprolactone (PCL) and varying their relative concentrations, PS/PCL, PS/PMMA and PCL/PMMA polymer demixed thin films were developed with nanoisland, nanoribbon and nanopit topographies. In the PS/PCL system, PS segregates to the air-polymer interface, with the lower solubility PCL preferring the substrate-polymer interface. In the PS/PMMA and PCL/PMMA systems, PMMA prefers the air-polymer interface due to its greater solubility and lower surface energy. The anti-adhesion efficacy of the demixed films were tested against Pseudomonas aeruginosa (PA14). PS/PCL and PCL/PMMA demixed films showed a significant reduction in cell counts adhered on their surfaces compared to pure polymer control films, while no reduction was observed in the counts adhered on PS/PMMA demixed films. While the specific morphology did not affect the adhesion, a relationship between bacterial cell and topographical surface feature size was apparent. If the surface feature was smaller than the cell, then an anti-adhesion effect was observed; if the surface feature was larger than the cell, then the bacteria preferred to adhere.

Antimicrobial Nitric Oxide Releasing Contact Lens Gels for the Treatment of Microbial Keratitis.

Microbial keratitis is a serious sight threatening infection affecting approximately two million individuals worldwide annually. While antibiotic eye drops remain the gold standard treatment for these infections, the significant problems associated with eye drop drug delivery and the alarming rise in antimicrobial resistance has meant that there is an urgent need to develop alternative treatments. In this work, a nitric oxide releasing contact lens gel displaying broad spectrum antimicrobial activity against two of the most common causative pathogens of microbial keratitis is described. The contact lens gel is composed of poly-ε-lysine (pεK) functionalized with nitric oxide (NO) releasing diazeniumdiolate moieties which enables the controlled and sustained release of bactericidal concentrations of NO at physiological pH over a period of 15 h. Diazeniumdiolate functionalization was confirmed by Fourier transform infrared (FTIR), and the concentration of NO released from the gels was determined by chemiluminescence. The bactericidal efficacy of the gels against Pseudomonas aeruginosa and Staphylococcus aureus was ascertained, and between 1 and 4 log reductions in bacterial populations were observed over 24 h. Additional cell cytotoxicity studies with human corneal epithelial cells (hCE-T) also demonstrated that the contact lens gels were not cytotoxic, suggesting that the developed technology could be a viable alternative treatment for microbial  keratitis.

The optimization and production of stable homogeneous amine enriched surfaces with characterized nanotopographical properties for enhanced osteoinduction of mesenchymal stem cells.

Silane modification has been proposed as a powerful biomaterial surface modification tool. This is the first comprehensive investigation into the effect of silane chain length on the resultant properties of -NH2 silane monolayers and the associated osteoinductive properties of the surface. A range of -NH2 presenting silanes, chain length 3-11, were introduced to glass coverslips and characterized using water contact angles, atomic force microscopy, X-ray photoelectron spectroscopy, and Ninhydrin assays. The ability of the variation in chain length to form a homogenous layer across the entirety of the surfaces was also assessed. The osteoinductive potential of the resultant surfaces was evaluated by real-time polymerase chain reaction, immunocytochemistry, and von Kossa staining. Control of surface chemistry and topography was directly associated with changes in chain length. This resulted in the identification of a specific, chain length 11 (CL11) which significantly increased the osteoinductive properties of the modified materials. Only CL11 surfaces had a highly regular nano-topography/roughness which resulted in the formation of an appetite-like layer on the surface that induced a significantly enhanced osteoinductive response (increased expression of osteocalcin, CBFA1, sclerostin, and the production of a calcified matrix) across the entirety of the surface. © 2018 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1862-1877, 2018.

Modified Mesoporous Silica Nanoparticles with a Dual Synergetic Antibacterial Effect.

Application of mesoporous silica nanoparticles (MSNs) as antifouling/antibacterial carriers is limited and specifically with a dual synergetic effect. In the present work, MSNs modified with quaternary ammonium salts (QASs) and loaded with the biocide Parmetol S15 were synthesized as functional fillers for antifouling/antibacterial coatings. From the family of the MSNs, MCM-48 was selected as a carrier because of its cubic pore structure, high surface area, and high specific pore volume. The QASs used for the surface modification of MCM-48 were dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride and dimethyltetradecyl[3-(triethoxysilyl)propyl]ammonium chloride. The QAS-modified MCM-48 reveals strong covalent bonds between the QAS and the surface of the nanoparticles. The surface functionalization was confirmed by Fourier transform infrared spectroscopy, thermogravimetric analysis, elemental analysis, and ζ-potential measurements. Additional loading of the QAS-modified MCM-48 with a commercially available biocide (Parmetol S15) resulted in a synergetic dual antibacterial/antifouling effect. Either loaded or unloaded QAS-modified MSNs exhibited high antibacterial performance confirming their dual activity. The QAS-modified MCM-48 loaded with the biocide Parmetol S15 killed all exposed bacteria after 3 h of incubation and presented 100% reduction at the antibacterial tests against Gram-negative and Gram-positive bacteria. Furthermore, the QAS-modified MCM-48 without Parmetol S15 presented 77-89% reduction against the exposed Gram-negative bacteria and 78-94% reduction against the exposed Gram-positive bacteria. In addition, the modified MCM-48 was mixed with coating formulations, and its antifouling performance was assessed in a field test trial in northern Red Sea. All synthesized paints presented significant antifouling properties after 5 months of exposure in real seawater conditions, and the dual antifouling effect of the nanoparticles was confirmed.

Nitric Oxide Releasing Polymeric Coatings for the Prevention of Biofilm Formation.

The ability of nitric oxide (NO)-releasing polymer coatings to prevent biofilm formation is described. NO-releasing coatings on (poly(ethylene terephthalate) (PET) and silicone elastomer (SE)) were fabricated using aminosilane precursors. Pristine PET and SE were oxygen plasma treated, followed by immobilisation of two aminosilane molecules: N-(3-(trimethoxysilyl)propyl)diethylenetriamine (DET3) and N-(3-trimethoxysilyl)propyl)aniline (PTMSPA). N-diazeniumdiolate nitric oxide donors were formed at the secondary amine sites on the aminosilane molecules producing NO-releasing polymeric coatings. The NO payload and release were controlled by the aminosilane precursor, as DET3 has two secondary amine sites and PTMSPA only one. The antibacterial efficacy of these coatings was tested using a clinical isolate of Pseudomonas aeruginosa (PA14). All NO-releasing coatings in this study were shown to significantly reduce P. aeruginosa adhesion over 24 h with the efficacy being a function of the aminosilane modification and the underlying substrate. These NO-releasing polymers demonstrate the potential and utility of this facile coating technique for preventing biofilms for indwelling medical devices.

Linker-free covalent immobilization of nisin using atmospheric pressure plasma induced grafting.

The linker-free covalent immobilization of polymers on surfaces has the potential to impart new properties and functions to surfaces for a wide range of applications. However, most current methods for the production of these surfaces involve multiple chemical steps and do not have a high degree of control over the chemical functionalities at the surface. A comprehensive study detailing the facile two-step covalent grafting of the antimicrobial peptide nisin onto polystyrene surfaces is reported. Functionalization is achieved using an atmospheric pressure plasma jet, and the reaction is monitored and compared with a standard wet chemical functionalization approach using a variety of analytical techniques. The reactive species produced by the atmospheric pressure plasma jet were analyzed by mass spectrometry and optical emission spectroscopy. The surface chemistry and topography of the functionalized surfaces were determined using contact angle measurements, Fourier infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy and atomic force microscopy respectively. Following surface analysis, the antimicrobial efficacy of the covalently grafted nisin against two major food borne pathogens (Staphylococcus aureus and Listeria monocytogenes) was assessed at two different pHs. The results demonstrated that a post-plasma treatment step after nisin deposition is required to covalently graft the peptide onto the surface. The covalent immobilization of nisin resulted in a significant reduction in bacterial counts within a short 30 minutes contact time. These surfaces were also significantly more antimicrobial compared to those prepared via a more traditional wet chemical approach indicating that the reported method could be a less expensive and less time consuming alternative.

Human Fetal Osteoblast Response on Poly(Methyl Methacrylate)/Polystyrene Demixed Thin Film Blends: Surface Chemistry Vs Topography Effects.

Recent advances in materials sciences have allowed for the development and fabrication of biomaterials that are capable of providing requisite cues to instigate cells to respond in a predictable fashion. We have developed a series of poly(methyl methacrylate)/polystyrene (PMMA/PS) polymer demixed thin films with nanotopographies ranging from nanoislands to nanopits to study the response of human fetal osteoblast cells (hFOBs). When PMMA was in excess in the blend composition, a nanoisland topography dominated, whereas a nanopit topography dominated when PS was in excess. PMMA was found to segregate to the top of the nanoisland morphology with PS preferring the substrate interface. To further ascertain the effects of surface chemistry vs topography, we plasma treated the polymer demixed films using an atmospheric pressure dielectric barrier discharge reactor to alter the surface chemistry. Our results have shown that hFOBs did not have an increased short-term cellular response on pristine polymer demixed surfaces. However, increasing the hydrophilicty/wettability of the surfaces by oxygen functionalization causes an increase in the cellular response. These results indicate that topography alone is not sufficient to induce a positive cellular response, but the underlying surface chemistry is also important in regulating cell function.

Protein, cell and bacterial response to atmospheric pressure plasma grafted hyaluronic acid on poly(methylmethacrylate).

Hyaluronic acid (HA) has been immobilised on poly(methyl methacrylate) (PMMA) surfaces using a novel dielectric barrier discharge (DBD) plasma process for the purposes of repelling protein, cellular and bacterial adhesion in the context of improving the performance of ophthalmic devices. Grafting was achieved by the following steps: (1) treatment of the PMMA with a DBD plasma operating at atmospheric pressure, (2) amine functionalisation of the activated polymer surface by exposure to a 3-aminopropyltrimethoxysilane (APTMS) linker molecule and (3) reaction of HA with the surface bound amine. The mechanism and effectiveness of the grafting process was verified by surface analysis. XPS data indicates that the APTMS linker molecule binds to PMMA via the Si-O chemistry and has the required pendant amine moiety. The carboxylic acid moiety on HA then binds with this -NH2 group via standard carbodiimide chemistry. ToF-SIMS confirms the presence of a coherent HA layer the microstructure of which is verified by AFM. The plasma grafted HA coating surfaces showed a pronounced decrease in protein and cellular adhesion when tested with bovine serum albumin and human corneal epithelial cells, respectively. The ability of these coatings to resist bacterial adhesion was established using Staphylococcus aureus NTC8325. Interestingly, the coatings did not repel bacterial adhesion, indicating that the mechanism of adhesion of bacterial cells is different to that for the surface interactions of mammalian cells. It is proposed that this difference is a consequence of the specific HA conformation that occurs under the conditions employed here. Hence, it is apparent that the microstructure/architecture of the HA coatings is an important factor in fabricating surfaces intended to repel proteins, mammalian and bacterial cells.

Human mesenchymal stem cell response to poly(ε-caprolactone/poly(methyl methacrylate) demixed thin films.

Advances in material sciences have enabled the fabrication of biomaterials which are able to provide the requisite cues to stimulate cells to behave in a specific way. Nanoscale surface topographies are well known to be able to positively influence cell-substrate interactions. This study reports on a novel series of poly(ε-caprolactone) PCL and poly(methyl methacrylate) demixed nanotopographic films as non-biological cell-stimulating cues. The topographic features observed ranged from nanoislands to nanopits. PMMA was observed to segregate to the air interface, while PCL preferred the substrate interface. Preliminary response of human mesenchymal stem cells to these surfaces indicated that the substrate with nanoisland topography has the potential to differentiate to osteogenic, chondrogenic and adipogenic lineages.

The role of enamel proteins in protecting mature human enamel against acidic environments: a double layer force spectroscopy study.

Characterisation of the electrostatic properties of dental enamel is important for understanding the interfacial processes that occur on a tooth surface and how these relate to the natural ability of our teeth to withstand chemical attack from the acids in many soft drinks. Whereas, the role of the mineral component of the tooth enamel in providing this resistance to acid erosion has been studied extensively, the influence of proteins that are also present within the structure is not well understood. In this paper, we report for the first time the use of double-layer force spectroscopy to directly measure electrostatic forces on as received and hydrazine-treated (deproteinated) enamel surfaces in solutions with different pH to determine how the enamel proteins influence acid erosion surface potential and surface charge of human dental enamel. The deproteination of the treated samples was confirmed by the loss of the amide bands (~1,300-1,700 cm(-1)) in the FTIR spectrum of the sample. The force characteristics observed were found to agree with the theory of electrical double layer interaction under the assumption of constant potential and allowed the surface charge per unit area to be determined for the two enamel surfaces. The values and, importantly, the sign of these adsorbed surface charges indicates that the protein content of dental enamel contributes significantly to the electrostatic double layer formation near the tooth surface and in doing so can buffer the apatite crystals against acid attack. Moreover, the electrostatic interactions within this layer are a driving factor for the mineral transfer from the tooth surface and the initial salivary pellicle formation.