To Örebro University

Periodontitis is an inflammatory condition affecting tooth-supportive structures, the periodontium. Gingival tissues respond to biofilm formation by initiating an inflammatory process. If left untreated, inflammation progresses to a non-reversible condition in susceptible individuals, causing degradation of the underlying bone structures, where teeth become mobile and tooth-loss eventually occurs. Periodontitis is very common, affecting about half of the adult population. The disease is not only a concern for oral health but is linked to many systemic conditions, such as cardiovascular disease, diabetes, rheumatoid arthritis, and Alzheimer’s.

The studies included in this thesis aim to develop new strategies for the diagnosis and treatment of periodontal disease. Current diagnostic practices rely on probing and x-ray examination. There is a need for more sophisticated diagnostic tools. We have developed different sensing strategies for detection of a key-pathogen in periodontitis, Porphyromonas gingivalis, and its secreted enzymes, gingipains. The proteolytic activity of this pathogen, which drives the destructive effects of the disease, can be detected at low levels by utilizing gold nanoparticles and nanoplasmonic sensing in different ways.

Treatment of periodontitis is basically limited to the removal of soft and hard biofilms. This requires significant resources, from patients as well as clinicians, and maintenance phase is often life-long. We have identified and tested the antibacterial effect of an antimicrobial peptide derived from Lactobacillus plantarum. This bacteriocin, plantaricin NC8αβ, has been shown to effectively inhibit P. gingivalis. The bacteriocin has been further optimized into a short peptide sequence, C5, which can rupture outer membrane vesicles from P. gingivalis. Taken together, these results suggest new sensing strategies for novel diagnostic tools for periodontal disease and define a promising antibacterial agent for periodontal treatment in the future.

Accurate, robust, and rapid diagnostics is the basis for all well-functioning healthcare. There is a large need in point-of-care biosensors to facilitate diagnosis and reduce the need for cumbersome laboratory equipment. Proteases are key virulence factors in periodontitis. Periodontal disease is very common and characterized by inflammation and infection in the tooth-supporting structures and is linked to many systemic diseases such as cardiovascular disease, diabetes, and Alzheimer's disease. Proteases present in periodontal disease, gingipains, are highly responsible for the disease onset and progression and are therefore a promising biomarker. Here we show a novel nanopore-based biosensor strategy for protease activity monitoring. Solid-state nanopores were modified with a proteolytic substrate, restricting the ionic current through the apertures of the nanopores. Protease can digest the proteolytic substrate thus enlarge the aperture and the ionic current. Trypsin was used as an initial model protease to investigate the performance of the sensor. We show that the solid-state nanoporebiosensor can detect trypsin with a limit of detection (LOD) of 0.005 ng/mL (0.2 pM). The detection system developed for the model enzyme was then applied to the detection of gingipains. The LOD for detection of gingipains was 1 ng/mL (0.02 nM), with a 27% recovery of the signal at 0.1 mu g/mL, indicating that the sensitivity and dynamic range are relevant for the clinical diagnosis of periodontitis. The generic detection of protease activity and high sensitivity make this a promising sensor technology for both diagnosis of periodontal disease and monitoring of other disease-related proteases.

Elastic fibers containing elastin play an important role in tendon functionality, but the knowledge on presence and function of elastin during tendon healing is limited. The aim of this study was to investigate elastin content and distribution in intact and healing Achilles tendons and to understand how elastin influence the viscoelastic properties of tendons. The right Achilles tendon was completely transected in 81 Sprague-Dawley rats. Elastin content was quantified in intact and healing tendons (7, 14, and 28 days post-surgery) and elastin distribution was visualized by immunohistochemistry at 14 days post-surgery. Degradation of elastin by elastase incubation was used to study the role of elastin on viscoelastic properties. Mechanical testing was either performed as a cyclic test (20x 10 N) or as a creep test. We found significantly higher levels of elastin in healing tendons at all time-points compared to intact tendons (4% in healing tendons 28 days post-surgery vs 2% in intact tendons). The elastin was more widely distributed throughout the extracellular matrix in the healing tendons in contrast to the intact tendon where the distribution was not so pronounced. Elastase incubation reduced the elastin levels by approximately 30% and led to a 40%-50% reduction in creep. This reduction was seen in both intact and healing tendons. Our results show that healing tendons contain more elastin and is more compliable than intact tendons. The role of elastin in tendon healing and tissue compliance indicates a protective role of elastic fibers to prevent re-injuries during early tendon healing. Plain Language Summary Tendons transfer high loads from muscles to bones during locomotion. They are primarily made by the protein collagen, a protein that provide strength to the tissues. Besides collagen, tendons also contain other building blocks such as, for example, elastic fibers. Elastic fibers contain elastin and elastin is important for the extensibility of the tendon. When a tendon is injured and ruptured the tissue heals through scar formation. This scar tissue is different from a normal intact tendon and it is important to understand how the tendons heal. Little is known about the presence and function of elastin during healing of tendon injuries. We have shown, in animal experiments, that healing tendons have higher amounts of elastin compared to intact tendons. The elastin is also spread throughout the tissue. When we reduced the levels of this protein, we discovered altered mechanical properties of the tendon. The healing tendon can normally extend quite a lot, but after elastin removal this extensibility was less obvious. The ability of the healing tissue to extend is probably important to protect the tendon from re-injuries during the first months after rupture. We therefore propose that the tendons heal with a large amount of elastin to prevent re-ruptures during early locomotion.

Periodontitis is an inflammatory oral disease that affects a large part of the adult population, causing significant costs and suffering. The key pathogen, Porphyromonas gingivalis, secretes gingipains, which are highly destructive proteases and the most important virulence factors in the pathogenesis of the disease. Currently, periodontitis is diagnosed mainly by mechanical manual probing and radiography, often when the disease has already progressed significantly. The possibilities of detecting gingipain activity in gingival fluid could enable early-stage diagnosis and facilitate treatment. Here, we describe a sensitive nanoparticle-based nanoplasmonic biosensor for the detection of the proteolytic activity of gingipains. Gold nanoparticles (AuNPs) were self-assembled as a submonolayer in multiwell plates and further modified with casein or IgG. The proteolytic degradation of the protein coating was tracked by monitoring the shift in the localized surface plasmon resonance (LSPR) peak position. The sensor performance was investigated using model systems with trypsin and purified gingipains (subtypes Kgp and RgpB) and further validated using supernatants from cultures of P. gingivalis. Proteolytic degradation by proteases in buffer results in a concentration- and time-dependent blueshift of the LSPR band of about 1-2 nm when using casein as a substrate. In bacterial supernatants, the degradation of the protein coating resulted in unspecific binding of proteins present in the complex sample matrix to the nanoparticles, which instead triggered a redshift of about 2 nm of the LSPR band. A significant LSPR shift was seen only in samples with gingipain activity. The sensor showed a limit of detection < 0.1 mu g/mL (4.3 nM), which is well below gingipain concentrations detected in severe chronic periodontitis cases (similar to 50 mu g/mL). This work shows the possibility of developing cost-effective nanoparticle-based biosensors for rapid detection of protease activity for chair-side periodontal diagnostics.

Conducting polymers are routinely used in optoelectronic biomaterials, but large polymer polydispersity and poor aqueous compatibility complicate integration with biomolecular templates and development of discrete and defined supramolecular complexes. Herein, we report on a chiro-optical hybrid material generated by the self-assembly of an anionic peptide and a chemically defined cationic pentameric thiophene in aqueous environment. The peptide acts as a stereochemical template for the thiophene and adopts an α-helical conformation upon association, inducing optical activity in the thiophene π-π* transition region. Theoretical calculations confirm the experimentally observed induced structural changes and indicate the importance of electrostatic interactions in the complex. The association process is also probed at the substrate-solvent interface using peptide-functionalized gold nanoparticles, indicating that the peptide can also act as a scaffold when immobilized, resulting in structurally well-defined supramolecular complexes. The hybrid complex could rapidly be assembled, and the kinetics of the formation could be monitored by utilizing the local surface plasmon resonance originating from the gold nanoparticles. We foresee that these findings will aid in designing novel hybrid materials and provide a possible route for the development of functional optoelectronic interfaces for both biomaterials and energy harvesting applications.