The role of bacterial stimuli in inflammation-driven bone formation.

Immune cells and their soluble factors regulate skeletal cells during normal bone regeneration and pathological bone formation. Bacterial infections can trigger immune responses that activate pro-osteogenic pathways, but these are usually overshadowed by osteolysis and concerns of systemic inflammation. The aim of this study was to determine whether the transient local inflammatory reaction to non-viable bacterial immune agonists could lead to favourable new bone formation. In a series of rabbit studies, as proof-of-concept, how tibial intramedullary injection of viable or killed bacterial species affected bone remodelling and new bone formation was determined. Application of killed bacteria led to considerable new bone formation after 4 weeks, without the prolonged systemic inflammation and exaggerated bone lysis seen with active infection. The osteo-immunomodulatory effects of various species of killed bacteria and the dose response relationship were subsequently screened in ectopically-implanted ceramic scaffolds. Histomorphometry after 8 weeks showed that a relatively low dose of killed bacteria enhanced ectopic bone induction. Moreover, lipoteichoic acid - the bacterial cell-wall derived toll-like-receptor (TLR)-2 activator - was identified as an osteo-stimulatory factor. Collectively, the data indicated that bacterial stimuli could be harnessed to stimulate osteogenesis, which occurs through a synergy with osteoinductive signals. This finding holds promise for the use of non-viable bacteria, bacterial antigens, or their simplified analogues as immuno-modulatory bone regenerating tools in bone biomaterials.

Growth factor-induced osteogenesis in a novel radiolucent bone chamber.

Treatment of large bone defects is currently performed using mainly autograft or allograft bone. There are important drawbacks to bone grafting, such as limited availability, donor site morbidity in the case of autograft and inferior performance of allografts. Therefore, there is a great need for a suitable bone graft substitute. In order to evaluate efficiently newly developed biomaterials and factors intended for orthopaedic surgery, the bone chamber is a very suitable model. To allow longitudinal investigation of bone growth with µCT, a new bone chamber made of radiolucent polyether ether ketone (PEEK) was developed and studied for its feasibility. Therefore, PEEK bone chambers were placed on rat tibiae, and filled with vehicle (Matrigel without growth factors, negative controls), with bone morphogenetic protein 2 (BMP-2, positive controls), or a mix of growth factors combining BMP-2, vascular endothelial growth factor and the chemokine stromal cell-derived factor 1α, all laden on gelatin microspheres for controlled release (combined growth factors). Growth factor presence led to a significant increase in bone formation after 8 weeks, which subsided after 12 weeks, underlining the importance of longitudinal analysis. We conclude that the PEEK-bone chamber is a suitable translational animal model to assess orthotopic bone formation in a longitudinal manner.

Full regeneration of segmental bone defects using porous titanium implants loaded with BMP-2 containing fibrin gels.

Regeneration of load-bearing segmental bone defects is a major challenge in trauma and orthopaedic surgery. The ideal bone graft substitute is a biomaterial that provides immediate mechanical stability, while stimulating bone regeneration to completely bridge defects over a short period. Therefore, selective laser melted porous titanium, designed and fine-tuned to tolerate full load-bearing, was filled with a physiologically concentrated fibrin gel loaded with bone morphogenetic protein-2 (BMP-2). This biomaterial was used to graft critical-sized segmental femoral bone defects in rats. As a control, porous titanium implants were either left empty or filled with a fibrin gels without BMP-2. We evaluated bone regeneration, bone quality and mechanical strength of grafted femora using in vivo and ex vivo µCT scanning, histology, and torsion testing. This biomaterial completely regenerated and bridged the critical-sized bone defects within eight weeks. After twelve weeks, femora were anatomically re-shaped and revealed open medullary cavities. More importantly, new bone was formed throughout the entire porous titanium implants and grafted femora regained more than their innate mechanical stability: torsional strength exceeded twice their original strength. In conclusion, combining porous titanium implants with a physiologically concentrated fibrin gels loaded with BMP-2 improved bone regeneration in load-bearing segmental defects. This material combination now awaits its evaluation in larger animal models to show its suitability for grafting load-bearing defects in trauma and orthopaedic surgery.

Clinical evaluation of a PHMB-impregnated biocellulose dressing on paediatric lacerations.

OBJECTIVE: To evaluate the clinical benefits, primarily tolerability and reduction in pain levels, associated with the use of a PHMB-impregnated biosynthetic cellulose dressing (Suprasorb X + PHMB) on paediatric heel lacerations. METHOD: These lacerations were caused when children, who were being transported on their parents' bicycles, got their heels trapped in the wheel spokes. Where these injuries just comprised skin contusion and laceration, treatment had previously comprised cleansing followed by application of conventional dressings and moist wound healing dressings. However, the high incidence of infection necessitated regular dressing changes, which caused parents and children stress and anxiety. This clinical evaluation assessed the benefits of a new treatment protocol, where the PHMB-impregnated biocellulose dressing was applied and left in situ until epithelialisation occurred. A cork splint was used for 3 days to prevent pes equinus and to let the ankle joint rest. Change in wound size (cm²), incidence of local infection, wound bed characteristics and pain levels (measured on a 0-10 paediatric pain scale) were assessed at 3-day intervals during the 14-day treatment period. Satisfaction with the dressing was also evaluated. RESULTS: Twenty children (mean age 5.6 years (± 1.33) were recruited into the study and included in the analysis. The mean baseline wound area was 8.60cm² (± 6.57). The mean time to complete wound closure was 12.95 days (± 7.69) with a mean total of 4.70 visits (± 1.56). The mean VAS pain score was 9.55 (± 0.69), compared with 0.15 (± 0.37) on day 14 (p<0.003). At the second visit (after 3 days) 17 of the 20 children were reported to be free of pain. No cases of local infection were noted. CONCLUSION: The dressing was found to be child and parent friendly. The evaluation also showed that it was well tolerated and achieved good healing outcome. It has now been incorporated into the clinic's treatment protocol for these wounds. CONFLICT OF INTEREST: None. The authors have no relevant financial interest in this article. All authors were involved in the critical revision of the manuscript for important intellectual content.

Antibacterial and immunogenic behavior of silver coatings on additively manufactured porous titanium.

Implant-associated infections (IAI) are often recurrent, expensive to treat, and associated with high rates of morbidity, if not mortality. We biofunctionalized the surface of additively manufactured volume-porous titanium implants using electrophoretic deposition (EPD) as a way to eliminate the peri-operative bacterial load and prevent IAI. Chitosan-based (Ch) coatings were incorporated with different concentrations of silver (Ag) nanoparticles or vancomycin. A full-scale in vitro and in vivo study was then performed to evaluate the antibacterial, immunogenic, and osteogenic activity of the developed implants. In vitro, Ch + vancomycin or Ch + Ag coatings completely eliminated, or reduced the number of planktonic and adherent Staphylococcus aureus by up to 4 orders of magnitude, respectively. In an in vivo tibia intramedullary implant model, Ch + Ag coatings caused no adverse immune or bone response under aseptic conditions. Following Staphylococcus aureus inoculation, Ch + vancomycin coatings reduced the implant infection rate as compared to chitosan-only coatings. Ch + Ag implants did not demonstrate antibacterial effects in vivo and even aggravated infection-mediated bone remodeling including increased osteoclast formation and inflammation-induced new bone formation. As an explanation for the poor antibacterial activity of Ch + Ag implants, it was found that antibacterial Ag concentrations were cytotoxic for neutrophils, and that non-toxic Ag concentrations diminished their phagocytic activity. This study shows the potential of EPD coating to biofunctionalize porous titanium implants with different antibacterial agents. Using this method, Ag-based coatings seem inferior to antibiotic coatings, as their adverse effects on the normal immune response could cancel the direct antibacterial effects of Ag nanoparticles. STATEMENT OF SIGNIFICANCE: Implant-associated infections (IAI) are a clinical, societal, and economical burden. Surface biofunctionalization approaches can render complex metal implants with strong local antibacterial action. The antibacterial effects of inorganic materials such as silver nanoparticles (Ag NPs) are often highlighted under very confined conditions in vitro. As a novelty, this study also reports the antibacterial, immunogenic, and osteogenic activity of Ag NP-coated additively-manufactured titanium in vivo. Importantly, it was found that the developed coatings could impair the normal function of neutrophils, the most important phagocytic cells protecting us from IAI. Not surprisingly, the Ag NP-based coatings were outperformed by an antibiotic-based coating. This emphasizes the importance of also targeting implant immune-modulatory functions in future coating strategies against IAI.

Fabrication of three-dimensional bioplotted hydrogel scaffolds for islets of Langerhans transplantation.

In clinical islet transplantation, allogeneic islets of Langerhans are transplanted into the portal vein of patients with type 1 diabetes, enabling the restoration of normoglycemia. After intra-hepatic transplantation several factors are involved in the decay in islet mass and function mainly caused by an immediate blood mediated inflammatory response, lack of vascularization, and allo- and autoimmunity. Bioengineered scaffolds can potentially provide an alternative extra-hepatic transplantation site for islets by improving nutrient diffusion and blood supply to the scaffold. This would ultimately result in enhanced islet viability and functionality compared to conventional intra portal transplantation. In this regard, the biomaterial choice, the three-dimensional (3D) shape and scaffold porosity are key parameters for an optimal construct design and, ultimately, transplantation outcome. We used 3D bioplotting for the fabrication of a 3D alginate-based porous scaffold as an extra-hepatic islet delivery system. In 3D-plotted alginate scaffolds the surface to volume ratio, and thus oxygen and nutrient transport, is increased compared to conventional bulk hydrogels. Several alginate mixtures have been tested for INS1E ß-cell viability. Alginate/gelatin mixtures resulted in high plotting performances, and satisfactory handling properties. INS1E ß-cells, human and mouse islets were successfully embedded in 3D-plotted constructs without affecting their morphology and viability, while preventing their aggregation. 3D plotted scaffolds could help in creating an alternative extra-hepatic transplantation site. In contrast to microcapsule embedding, in 3D plotted scaffold islets are confined in one location and blood vessels can grow into the pores of the construct, in closer contact to the embedded tissue. Once revascularization has occurred, the functionality is fully restored upon degradation of the scaffold.