Bone formation by heterodimers through non-viral gene delivery of BMP-2/6 and BMP-2/7.

Non-viral gene delivery is a safe technique to release sustained physiologic dosages of bone morphogenetic protein (BMP). Co-delivery of multiple BMPs can result in the formation of more potent BMP heterodimers. In this study, non-viral co-delivery of BMP-2/6 and BMP-2/7, as a mean to produce heterodimers, was assessed. Goat MSCs were non-virally transfected with plasmid DNA encoding BMP isoforms (pBMP) known to be relevant for osteogenesis: BMP-2, -6 or -7. As a result, BMP-2, -6 and -7 were produced and detectable for up to 14 d and their combined delivery (pBMP-2 with pBMP-6 or pBMP-7) was used to create BMP-2/6 and BM-2/7 heterodimers. Formation and secretion of the heterodimer proteins was validated by sandwich enzyme-linked immunosorbent assay (ELISA). Produced BMPs and heterodimers were biologically active, as confirmed by differentiation of reporter cells and MSCs. To assess bone formation, transfected MSCs were seeded on to ceramic scaffolds and implanted subcutaneously into nude mice. Bone formation was significantly enhanced in the pBMP-2/6 condition and a trend for more bone formation was observed in the pBMP-2/7 and pBMP-6 homodimer condition. No bone was found in the pBMP-2, pBMP-7 or control condition. In conclusion, simultaneous delivery of pBMP-2 with pBMP-6 or -7 resulted in the production of heterodimers that were beneficial for bone formation as compared to BMP homodimers. Combination of BMP sequences could reduce the need for high BMP protein dosages and might enhance prolonged availability of the growth factors.

Local induction of inflammation affects bone formation.

To explore the influence of inflammatory processes on bone formation, we applied a new in vivo screening model. Confined biological pockets were first created in rabbits as a response to implanted bone cement discs. These biomembrane pockets were subsequently used to study the effects of inflammatory stimuli on ectopic bone formation within biphasic calcium phosphate (BCP) constructs loaded with TNF-α, lipopolysaccharide (LPS) or lipoteichoic acid (LTA), all with or without bone morphogenetic protein (BMP)-2. Analysis of bone formation after 12 weeks demonstrated that the inflammatory mediators were not bone-inductive in combination with the BCP alone, but inhibited or enhanced BMP-induced bone formation. LPS was associated with a strong inhibition of bone formation by BMP-2, while LTA and TNF-α showed a positive interaction with BMP-2. Since the biomembrane pockets did not interfere with bone formation and prevented the leakage of pro-inflammatory compounds to the surrounding tissue, the biomembrane model can be used for in vivo approaches to study local inflammation in conjunction with new bone formation. Using this model, it was shown that the modulation of the inflammatory response could be beneficial or detrimental to the subsequent bone formation process. The co-delivery of inflammatory factors and bone-related growth factors should be further explored as a strategy to enhance the bone-forming efficacy of bone substitutes.

Antibacterial Behavior of Additively Manufactured Porous Titanium with Nanotubular Surfaces Releasing Silver Ions.

Additive manufacturing (3D printing) has enabled fabrication of geometrically complex and fully interconnected porous biomaterials with huge surface areas that could be used for biofunctionalization to achieve multifunctional biomaterials. Covering the huge surface area of such porous titanium with nanotubes has been already shown to result in improved bone regeneration performance and implant fixation. In this study, we loaded TiO2 nanotubes with silver antimicrobial agents to equip them with an additional biofunctionality, i.e., antimicrobial behavior. An optimized anodizing protocol was used to create nanotubes on the entire surface area of direct metal printed porous titanium scaffolds. The nanotubes were then loaded by soaking them in three different concentrations (i.e., 0.02, 0.1, and 0.5 M) of AgNO3 solution. The antimicrobial behavior and cell viability of the developed biomaterials were assessed. As far as the early time points (i.e., up to 1 day) are concerned, the biomaterials were found to be extremely effective in preventing biofilm formation and decreasing the number of planktonic bacteria particularly for the middle and high concentrations of silver ions. Interestingly, nanotubes not loaded with antimicrobial agents also showed significantly smaller numbers of adherent bacteria at day 1, which may be attributed to the bactericidal effect of high aspect ratio nanotopographies. The specimens with the highest concentrations of antimicrobial agents adversely affected cell viability at day 1, but this effect is expected to decrease or disappear in the following days as the rate of release of silver ions was observed to markedly decrease within the next few days. The antimicrobial effects of the biomaterials, particularly the ones with the middle and high concentrations of antimicrobial agents, continued until 2 weeks. The potency of the developed biomaterials in decreasing the number of planktonic bacteria and hindering the formation of biofilms make them promising candidates for combating peri-operative implant-associated infections.