Experimental Studies on Non-Metallic Composite Bone Implants With a Special Reference to Staphylococcal Biofilm Infection

Non-metallic implants made of bioresorbable or biostable synthetic polymers are attractive options in many surgical procedures, ranging from bioresorbable suture anchors of arthroscopic surgery to reconstructive skull implants made of biostable fiber-reinforced composites. Among other benefits, non-metallic implants produce less interference in imaging. Bioresorbable polymer implants may be true multifunctional, serving as osteoconductive scaffolds and as matrices for simultaneous delivery of bone enhancement agents. As a major advantage for loading conditions, mechanical properties of biostable fiber-reinforced composites can be matched with those of the bone. Unsolved problems of these biomaterials are related to the risk of staphylococcal biofilm infections and to the low osteoconductivity of contemporary bioresorbable composite implants. This thesis was focused on the research and development of a multifunctional implant model with enhanced osteoconductivity and low susceptibility to infection. In addition, the experimental models for assessment, diagnostics and prophylaxis of biomaterial-related infections were established. The first experiment (Study I) established an in vitro method for simultaneous evaluation of calcium phosphate and biofilm formation on bisphenol-Aglycidyldimethacrylate and triethylenglycoldimethacrylate (BisGMA-TEGDMA) thermosets with different content of bioactive glass 45S5. The second experiment (Study II) showed no significant difference in osteointegration of nanostructured and microsized polylactide-co-glycolide/β-tricalcium phosphate (PLGA /β-TCP) composites in a minipig model. The third experiment (Study III) demonstrated that positron emission tomography (PET) imaging with the novel 68Ga labelled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) CD33 related sialic-acid immunoglobulin like lectins (Siglec-9) tracer was able to detect inflammatory response to S. epidermidis and S. aureus peri-implant infections in an intraosseous polytetrafluoroethylene catheter model. In the fourth experiment (Study IV), BisGMATEGDMA thermosets coated with lactose-modified chitosan (Chitlac) and silver nanoparticles exhibited antibacterial activity against S. aureus and P. aeruginosa strains in an in vitro biofilm model and showed in vivo biocompatibility in a minipig model. In the last experiment (Study V), a selective androgen modulator (SARM) released from a poly(lactide)-co-ε-caprolactone (PLCL) polymer matrix failed to produce a dose-dependent enhancement of peri-implant osteogenesis in a bone marrow ablation model.

Fiber-reinforced Composite as Oral Implant Material. Experimental studies of glass fiber and bioactive glass in vitro and in vivo

Fiber-reinforced composite as oral implant material: Experimental studies of glass fiber and bioactive glass in vitro and in vivo Department of Prosthetic Dentistry and Biomaterials Science, Institute of Dentistry, University of Turku, Turku, Finland 2008. Biocompatibility and mechanical properties are important variables that need to be determined when new materials are considered for medical implants. Special emphasis was placed on these characteristics in the present work, which aimed to investigate the potential of fiber-reinforced composite (FRC) material as an oral implant. Furthermore, the purpose of this study was to explore the effect of bioactive glass (BAG) on osseointegration of FRC implants. The biocompatibility and mechanical properties of FRC implants were studied both in vitro and in vivo. The mechanical properties of the bulk FRC implant were tested with a cantilever bending test, torsional test and push-out test. The biocompatibility was first evaluated with osteoblast cells cultured on FRC substrates. Bone bonding was determined with the mechanical push-out test and histological as well as histomorplanimetric evaluation. Implant surface was characterized with SEM and EDS analysis. The results of these studies showed that FRC implants can withstand the static load values comparably to titanium. Threaded FRC implants had significantly higher push-out strength than the threaded titanium implants. Cell culture study revealed no cytotoxic effect of FRC materials on the osteoblast-like-cells. Addition of BAG particles enhanced cell proliferation and mineralization of the FRC substrates The in vivo study showed that FRC implants can withstand static loading until failure without fracture. The results also suggest that the FRC implant is biocompatible in bone. The biological behavior of FRC was comparable to that of titanium after 4 and 12 weeks of implantation. Furthermore, addition of BAG to FRC implant increases peri-implant osteogenesis and bone maturation.

PET Imaging of Osteomyelitis. Feasibility of 18F-FDG, 68Ga-Chloride and 68Ga-DOTAVAP-P1 Tracers in Staphylococcal Bone Infections

Due to technical restrictions of the database system the title of the thesis does not show corretly on this page. Numbers in the title are in superscript. Please see the PDF-file for correct title. ---- Osteomyelitis is a progressive inflammatory disease of bone and bone marrow that results in bone destruction due to an infective microorganism, most frequently Staphylococcus aureus. Orthopaedic concern relates to the need for reconstructive and trauma-related surgical procedures in the fast grow¬ing population of fragile, aged patients, who have an increased susceptibility to surgical site infections. Depending on the type of osteomyelitis, infection may be acute or a slowly progressing, low-grade infection. Peri-implant infections lead to implant loosening. The emerging antibiotic resistance of com¬mon pathogens further complicates the situation. With current imaging methods, significant limitations exist in the diagnosing of osteomyelitis and implant-related infections. Positron emission tomography (PET) with a glucose analogue, 18F-fluoro¬deoxyglucose (18F-FDG), seems to facilitate a more accurate diagnosis of chronic osteomyelitis. The method is based on the increased glucose consumption of activated inflammatory cells. Unfortunately, 18F-FDG accumulates also in sterile inflammation regions and causes false-positive findings, for exam¬ple, due to post-operative healing processes. Therefore, there is a clinical need for new, more infection-specific tracers. In addition, it is still unknown why 18F-FDG PET imaging is less accurate in the detec¬tion of periprosthetic joint infections, most frequently due to Staphylococcus epidermidis. This doctoral thesis focused on testing novel PET tracers (68Ga-chloride and 68Ga-DOTAVAP-P1) for early detections of bone infections and evaluated the role of pathogen-related factors in the appli¬cations of 18F-FDG PET in the diagnostics of bone infections. For preclinical models of S. epidermidis and S. aureus bone/implant infections, the significance of the causative pathogen was studied with respect to 18F-FDG uptake. In a retrospective analysis of patients with confirmed bone infections, the significance of the presence or absence of positive bacterial cultures on 18F-FDG uptake was evalu¬ated. 18F-FDG and 68Ga-chloride resulted in a similar uptake in S. aureus osteomyelitic bones. However, 68Ga-chloride did not show uptake in healing bones, and therefore it may be a more-specific tracer in the early post-operative or post-traumatic phase. 68Ga-DOTAVAP-P1, a novel synthetic peptide bind¬ing to vascular adhesion protein 1 (VAP-1), was able to detect the phase of inflammation in healing bones, but the uptake of the tracer was elevated also in osteomyelitis. Low-grade peri-implant infec¬tions due to S. epidermidis were characterized by a low uptake of 18F-FDG, which reflects the virulence of the causative pathogen and the degree of leukocyte infiltration. In the clinical study, no relationship was found between the level of 18F-FDG uptake and the presence of positive or negative bacterial cul¬tures. Thus 18F-FDG PET may help to confirm metabolically active infection process in patients with culture-negative, histologically confirmed, low-grade osteomyelitis.

Viral and cellular modulation of virus-induced apoptosis in experimental infection models

The aim of this study was to investigate herpes simplex virus type 1 (HSV-1)- and measles virus (MV)-induced cell death. HSV-1 with deletion in genes encoding infected cell protein (ICP)4 and protein kinase Us3 (d120) induced apoptosis and cathepsin activation in epithelial (HEp-2) and monocytic (U937) cells. Inhibition of cathepsin activity decreased the amount of d120-induced apoptosis indicating that d120-induced apoptosis could be cathepsin-mediated. Also, HSV-1 infection increased caspase activation suggesting that d120-induced apoptosis is probably caspase-mediated. Cystatin treatment decreased the activity of cathepsins and the replication of HSV-1 indicating that cathepsins contribute to HSV-1 infection. Interestingly, d120 induced also necroptosis in monocytic cells. This is the first report on necroptosis in HSV-1- infected cells. MV induced apoptosis in uninfected bystander T lymphocytes, probably via interaction of MV-infected monocytes with uninfected lymphocytes. The expression of death receptor Fas was clearly increased on the surface of lymphocytes. The number of apoptotic cells and the activation of cathepsins and caspases were increased in MVinfected U937 cells suggesting that MV-induced apoptosis could be cathepsin- and caspase-mediated. Cystatin treatment inhibited cathepsin activities but not MV-induced apoptosis. Besides HSV-1-induced apoptosis, innate immune responses were studied in HSV-1-infection. HSV-1 viruses with either ICP4 and Us3, or Us3 deletion only, increased the expression of Toll-like receptor (TLR)3 and stimulated its downstream pathways leading to increased expression of type I interferon gene and to functional interferons. These findings suggest that besides controlling apoptosis, HSV-1 ICP4 and Us3 genes are involved in the control of TLR3 response in infected cell.