Therapeutic pathogen recognition receptor (PRR) ligands are reaching clinical practice following their ability to skew the immune response in a specific direction. We investigated the effects of various therapeutic PRR ligands on bone cell differentiation and inflammation. Following stimu-lation, alkaline phosphatase (ALP) activity (Day 10), osteocalcin, osteonectin expression (Day 14), and calcium deposition (Day 21) were quantified in bone marrow-derived human mesenchymal stem cells (hMSCs). The osteoclastogenic response was determined by measuring tartrate-resistant acid phosphate (TRAP) activity in human monocytes. TNF-α, IL-6, IL-8, and IL-10 expressions were measured by enzyme-linked immunosorbent assay as an indicator of the ligands’ inflammatory properties. We found that nucleic acid-based ligands Poly(I:C) and CpG ODN C increased early ALP activity in hMSCs by 4-fold without affecting osteoclast formation. These ligands did not enhance expression of the other, late osteogenic markers. MPLA, Curdlan, and Pam3CSK4 did not affect osteogenic differentiation, but inhibited TRAP activity in monocytes, which was associated with increased expression of all measured cytokines. Nucleic acid-based ligands are identified as the most promising osteo-immunomodulators, as they favor early osteogenic differentiation without inducing an exaggerated immune-cell mediated response or interfering in osteoclastogenesis and thus can be potentially harnessed for multifunctional coatings for bone biomaterials.

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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.

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The main challenge in bone morphogenic protein 2 (BMP-2)-based application lies in finding strategies that prolong its effective period as it has a short biological half-life. Several BMP-2 release profiles have shown to enhance bone formation at various application sites. However, it remains to be determined which BMP-2 release profile best augments bone formation and whether this effect is location dependent. Therefore, the aim of this study was to investigate the effect of BMP-2 release from oligo[(polyethylene glycol) fumarate] bis(2-(methacryloyloxy)ethyl) phosphate (OPF-BP) composites on the osteoinductive efficacy at ectopic versus orthotopic application. By varying the BMP-2 loading method, three different OPF-BP composites were created with varied release profiles. The composites were compared with unloaded OPF-BP as negative control, and to the clinically used Infuse ® absorbable collagen sponge (ACS) as positive control. Bone formation was assessed by microcomputed tomography after 9 weeks of subcutaneous implantation and 3, 6, and 9 weeks of orthotopic implantation in rats (n = 48). Whereas a BMP-2 burst release of >49% generated significantly more bone compared with sustained release (burst release <30%) at the subcutaneous implantation site, differential release did not affect bone formation at the orthotopic site. Furthermore, all BMP-2 containing OPF-BP composites showed significantly more bone formation compared with ACS in the orthotopic implantation site. In conclusion, this study clearly shows that the osteoinductive effect of different BMP-2 release profiles is location dependent. In addition, more bone formation in OPF-BP compared with ACS at both application sites emphasizes the role of biomaterials as a scaffold to achieve proper bone tissue formation. The main challenge in bone morphogenic protein 2 (BMP-2)-based application lies in finding strategies to prolong its biologic activity as it has a short biological half-life. The present study uses a phosphate-modified oligo[(polyethylene glycol) fumarate] hydrogel that can be tuned to achieve differential release profiles of biologically active BMP-2 release. We demonstrate that this platform outperforms Infuse ® , currently used in the clinic and that the osteoinductive effect of BMP-2 is location dependent. Altogether, this study stresses the importance of evaluating efficacy of bone tissue engineering strategies at an orthotopic location rather than subcutaneously. Even more so, it emphasizes the role of biomaterials as a scaffold to achieve proper bone tissue engineering. @en

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.

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A model is needed to study the effectiveness of different anti-bacterial coatings on complex metal implants in a bone environment. This article shares data on the design of porous titanium implants for intramedullary implantation in the proximal rat tibia. The implant length, diameter and porosity were optimized after testing on cadaveric specimens. This article shares data on which parameters are critical to establish a chronic implant infection in Sprague Dawley rats when using the new implant design. To this end, different strains of Staphylococcus aureus and inoculation doses were investigated.@en

Pathologic conditions associated with bone formation can serve as models to identify bone-promoting mediators. The inflammatory response to bacterial infections generally leads to osteolysis and impaired bone healing, but
paradoxically, it can also have pro-osteogenic effects. As a potential model to investigate pro-osteogenic stimuli, this study characterizes the bone formation in an established rabbit tibia model of periprosthetic infection. Our hypothesis
was that the infection with Staphylococcus aureus (S. aureus) correlates with bone formation as a response to local inflammation. Fluorochromes showed excessive subperiosteal bone formation in infected tibiae, starting the first
week and continuing throughout the study period. Despite the observed cortical lysis on micro-CT after 28 days, infection resulted in a twofold higher bone volume in the proximal tibiae compared to uninfected controls. The
ipsilateral fibulae, nor the contralateral fibulae or tibiae were affected by infection. Next, we sought to confine the cause of stimulated bone formation to the isolated S. aureus cell wall. In absence of virulent bacterial infection, the S.
aureus cell wall extract induced bone in a more favorable way without cortical lysis. This suggests that the sterile inflammatory reaction to bacterial antigens may be harnessed for bone regenerative purposes. Future investigations
in this rabbit tibia model can lead to further identification of effective stimuli for clinical application.@en

Off the shelf availability in large quantities, drug delivery functionality, and modifiable chemistry and mechanical properties makes synthetic polymers highly suitable candidates for bone grafting. However, most synthetic polymers lack the ability to support cell attachment, proliferation, migration and differentiation and ultimately tissue formation. Incorporating anionic peptides into the polymer that mimic acidic proteins, which contribute to biomineralization and cellular attachment, could enhance bone formation. Therefore, this study investigates the effect of a phosphate functional group on osteoconductivity and BMP-2 induced bone formation in an injectable and biodegradable oligo[(polyethylene glycol) fumarate] (OPF) hydrogel. Three types of OPF hydrogels were fabricated using 0%, 20% or 40% Bis(2-(methacryloyloxy)ethyl) phosphate (BP) creating unmodified OPF-noBP and phosphate modified OPF-BP20 and OPF-BP40, respectively. To account for the osteoinductive effect of various BMP-2 release profiles, two different release profiles (i.e. different ratios of burst and sustained release) were obtained by varying the BMP-2 loading method. To investigate the osteoconductive effect of phosphate modification, unloaded OPF composites were assessed for bone formation in a bone defect model after 3, 6 and 9 weeks. To determine the effect of the hydrogel phosphate modification on BMP-2 induced bone formation, BMP-2 loaded OPF composites with differential BMP-2 release were analyzed after 9 weeks of subcutaneous implantation in rats. The phosphate modified OPF hydrogels (OPF-BP20, OPF-BP40) generated significantly more bone in an orthotopic defect compared to the unmodified hydrogel (OPF-noBP). Furthermore, the phosphate functionalized surface enhanced BMP-2 induced ectopic bone formation regardless of the BMP-2 release profile. In conclusion, this study clearly shows that phosphate functional groups improve the osteoconductive properties of OPF and enhanced BMP-2 induced bone formation. Therefore, functionalizing hydrogels with phosphate groups by crosslinking monomers into the hydrogel matrix could provide a valuable method for improving polymer characteristics and hold great promise for bone tissue engineering.@en

Bone morphogenetic protein-2 (BMP-2) gene delivery has shown to induce bone formation in vivo in cell-based tissue engineering. In addition, the chemoattractant stromal cell-derived factor-1α (SDF-1α, also known as CXCL12) is known to recruit multipotent stromal cells towards its release site where it enhances vascularisation and possibly contributes to osteogenic differentiation. To investigate potential cooperative behaviour for bone formation, we investigated combined release of BMP-2 and SDF-1α on ectopic bone formation in mice. Multipotent stromal cellseeded and cell-free constructs with BMP-2 plasmid DNA and /or SDF-1α loaded onto gelatin microparticles, were implanted subcutaneously in mice for a period of 6 weeks. Histological analysis and histomorphometry revealed that the onset of bone formation and the formed bone volume were both enhanced by the combination of BMP-2 and SDF-1α compared to controls in cell-seeded constructs. Samples without seeded multipotent stromal cells failed to induce any bone formation. We conclude that the addition of stromal cellderived factor-1α to a cell-seeded alginate based bone morphogenetic protein-2 plasmid DNA construct has an additive effect on bone formation and can be considered a promising combination for bone regeneration.@en

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. @en