Nanoscale topography increases the bioactivity of a material and stimulates specific responses (third generation biomaterial properties) at the molecular level upon first generation (bioinert) or second generation (bioresorbable or bioactive) biomaterials. We developed a technique (based upon the effects of nanoscale topography) that facilitated the in vitro expansion of bone graft for subsequent implantation and investigated the optimal conditions for growing new mineralised bone in vitro. Two topographies (nanopits and nanoislands) were embossed into the bioresorbable polymer Polycaprolactone (PCL). Three dimensional cell culture was performed using double-sided embossing of substrates, seeding of both sides, and vertical positioning of substrates. The effect of Hydroxyapatite, and chemical stimulation were also examined. Human bone marrow was harvested from hip arthroplasty patients, the mesenchymal stem cells culture expanded and used for cellular analysis of substrate bioactivity. The cell line specificity and osteogenic behaviour was demonstrated through immunohistochemistry, confirmed by real-time PCR and quantitative PCR. Mineralisation was demonstrated using alizarin red staining. Results showed that the osteoinduction was optimally conferred by the presence of nanotopography, and also by the incorporation of hydroxyapatite (HA) into the PCL. The nanopit topography and HA were both superior to the use of BMP2 in the production of mineralised bone tissue. The protocol from shim production to bone marrow harvesting and vertical cell culture on nanoembossed HaPCL has been shown to be reproducible and potentially applicable to economical larger scale production

Prosthetic joint infection is one of the most challenging complications of joint alloplasty and the diagnosis remains difficult. The aim of the study was to investigate the bacterial flora in surgical samples from 22 prosthetic patients using a panel of culture-independent molecular methods including broad range 16S rRNA gene PCR, cloning, sequencing, phylogeny, quantitative PCR (qPCR), and fluorescence in situ hybridization (FISH). Concomitant samples were cultured by standard methods. Molecular methods detected presence of bacteria in samples from 12 of 22 patients. Using clone libraries a total of 40 different bacterial species were identified including known pathogens and species not previously described in association with prosthetic joint infections. The predominant species were Propionibacterium acnes and Staphylococcus epidermidis; polymicrobial infections were found in 9 patients. Culture-based methods showed bacterial growth in 8 cases with the predominant species being S. epidermidis. Neither anaerobic bacteria (including P. acnes) nor any of the species not previously described in implant infections were isolated. Additionally, 7 of the 8 culture positive cases were monomicrobial. Overall, the results of culture-based and molecular methods showed concordance in 11 cases (hereof 9 negative by both methods) and discrepancy in 6 cases. In the remaining 5 cases, culture-based methods identified only one species or a group of bacteria (e.g., coagulase negative staphylococci or coryneform rods), while culture-independent molecular methods were able to detect several distinct bacterial species including a species within the group identified by culture. A qPCR assay was developed to assess the abundance of Propionibacterium while S. aureus was quantified by a published S. aureus qPCR assay. These quantifications confirmed the findings from the clone library approach and showed the potential of qPCR for fast detection of bacteria in orthopedic samples. Additionally, both single cells and microcolonies were visualized using FISH and confocal scanning laser microscopy. In conclusion, the molecular methods detected a more diverse bacterial flora in prosthetic joint infections than revealed by standard culture-based methods, and polymicrobial infections were more frequently observed. The pathogenesis of these microorganisms and their role in implant-associated infections needs to be determined