In vitro and in vivo evaluation of chitosan scaffolds combined with simvastatin-loaded nanoparticles for guided bone regeneration.

The objective of this study was to fabricate and characterize chitosan combined with different amounts of simvastatin-loaded nanoparticles and to investigate their potential for guided bone regeneration in vitro and in vivo. Different SIM-CSN formulations were combined into a chitosan scaffold (SIM-CSNs-S), and the morphology, simvastatin release profile, and effect on cell proliferation and differentiation were investigated. For in vivo experiments, ectopic osteogenesis and the critical-size cranial defect model in SD rats were chosen to evaluate bone regeneration potential. All three SIM-CSNs-S formulations had a porous structure and exhibited sustained simvastatin release. CSNs-S showed excellent degradation and biocompatibility characteristics. The 4 mg SIM-CSNs-S formulation stimulated higher BMSC ALP activity levels, demonstrated significantly earlier collagen enhancement, and led to faster bone regeneration than the other formulations. SIM-CSNs-S should have a significant effect on bone regeneration.

Topographical cues of direct metal laser sintering titanium surfaces facilitate osteogenic differentiation of bone marrow mesenchymal stem cells through epigenetic regulation.

OBJECTIVES: To investigate the role of hierarchical micro/nanoscale topography of direct metal laser sintering (DMLS) titanium surfaces in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), as well as the possible underlying epigenetic mechanism. MATERIALS AND METHODS: Three groups of titanium specimens were prepared, including DMLS group, sandblasted, large-grit, acid-etched (SLA) group and smooth titanium (Ti) group. BMSCs were cultured on discs followed by surface characterization. Cell adhesion and proliferation were examined by SEM and CCK-8 assay, while osteogenic-related gene expression was detected by real-time RT-PCR. Immunofluorescence, western blotting and in vivo study were also performed to evaluate the potential for osteogenic induction of materials. In addition, to investigate the underlying epigenetic mechanisms, immunofluorescence and western blotting were performed to evaluate the global level of H3K4me3 during osteogenesis. The H3K4me3 and H3K27me3 levels at the promoter area of the osteogenic gene Runx2 were detected by ChIP assay. RESULTS: The DMLS surface exhibits greater protein adsorption ability and shows better cell adhesion performance than SLA and Ti surfaces. Moreover, both in vitro and in vivo studies demonstrated that the DMLS surface is more favourable for the osteogenic differentiation of BMSCs than SLA and Ti surfaces. Accordingly, osteogenesis-associated gene expression in BMSCs is efficiently induced by a rapid H3K27 demethylation and increase in H3K4me3 levels at gene promoters upon osteogenic differentiation on DMLS titanium surface. CONCLUSIONS: Topographical cues of DMLS surfaces have greater potential for the induction of osteogenic differentiation of BMSCs than SLA and Ti surfaces both in vitro and in vivo. A potential epigenetic mechanism is that the appropriate topography allows rapid H3K27 demethylation and an increased H3K4me3 level at the promoter region of osteogenesis-associated genes during the osteogenic differentiation of BMSCs.

Regenerating infected bone defects with osteocompatible microspheres possessing antibacterial activity.

Treatment of infected bone defects still remains a formidable clinical challenge, and the design of bone implants with both anti-bacterial activity and -osteogenesis effects is nowadays regarded as a powerful strategy for infection control and bone healing. In the present study, bioresorbable porous-structured microspheres were fabricated from an amphiphilic block copolymer composed of poly(l-lactide) and poly(ethyl glycol) blocks. After being surface coated with mussel-inspired polydopamine, the microspheres were loaded with nanosilver via the reduction of silver nitrate and apatite via biomineralization in sequence. At optimized loading amounts, the nanosilver-loaded microspheres showed no unfavorable effects on the proliferation and differentiation of bone marrow mesenchymal stem cells despite preserving strong antibacterial activity in in vitro evaluations. For the critical-sized defects (φ = 8 mm) in the rat cranium that was pre-infected with Staphylococcus aureus, the filling of the dual-purpose microspheres demonstrated an effective way to kill bacteria in vivo, and in the meantime, it promoted new bone formation efficiently alongside the degradation of microspheres. Thus, the results suggested that bioresorbable microspheres with both osteoconductive and antibacterial activities were a good choice for treating infected bone defects.

Surface modification of titanium with hydroxyapatite layer induced by phase-transited lysozyme coating.

Surface modification of titanium with a hydroxyapatite (HAP) coating can improve the bioactivity of pristine titanium. The traditional techniques for coating HAP on titanium involve nonmild treatments using strong bases or acids or high temperatures. In this study, the coating of HAP was carried out by a novel methodology called phase-transited lysozyme-assisted hydroxyapatite formation (PAH); in this process of biomimetic mineralization, the abundant functional carboxyl groups of phase-transited lysozyme (PTL) were responsible for the nucleation of HAP crystals by concentrating Ca2+ ions at the interface between PTL and CaCl2 solution and for the subsequent growth of HAP crystals occurring in simulated body fluid (SBF). In vitro and in vivo experiments verified that the surface of titanium modified with the HAP/PTL-Ti multilayer was endowed with improved biocompatibility and osteoinductivity compared with those of pristine titanium. Therefore, PAH is a simple, rapid, low-cost and green process for the surface modification of titanium with an HAP coating and thus will be a promising methodology for the surface modification of titanium implants.

Oriented and Ordered Biomimetic Remineralization of the Surface of Demineralized Dental Enamel Using HAP@ACP Nanoparticles Guided by Glycine.

Achieving oriented and ordered remineralization on the surface of demineralized dental enamel, thereby restoring the satisfactory mechanical properties approaching those of sound enamel, is still a challenge for dentists. To mimic the natural biomineralization approach for enamel remineralization, the biological process of enamel development proteins, such as amelogenin, was simulated in this study. In this work, carboxymethyl chitosan (CMC) conjugated with alendronate (ALN) was applied to stabilize amorphous calcium phosphate (ACP) to form CMC/ACP nanoparticles. Sodium hypochlorite (NaClO) functioned as the protease which decompose amelogenin in vivo to degrade the CMC-ALN matrix and generate HAP@ACP core-shell nanoparticles. Finally, when guided by 10 mM glycine (Gly), HAP@ACP nanoparticles can arrange orderly and subsequently transform from an amorphous phase to well-ordered rod-like apatite crystals to achieve oriented and ordered biomimetic remineralization on acid-etched enamel surfaces. This biomimetic remineralization process is achieved through the oriented attachment (OA) of nanoparticles based on non-classical crystallization theory. These results indicate that finding and developing analogues of natural proteins such as amelogenin involved in the biomineralization by natural macromolecular polymers and imitating the process of biomineralization would be an effective strategy for enamel remineralization. Furthermore, this method represents a promising method for the management of early caries in minimal invasive dentistry (MID).

Rapid biomimetic remineralization of the demineralized enamel surface using nano-particles of amorphous calcium phosphate guided by chimaeric peptides.

OBJECTIVE: The objective of this study was to develop a rapid and effective method to remineralize human carious-like enamel using chimaeric peptide-mediated nanocomplexes of carboxymethyl chitosan/amorphous calcium phosphate (CMC/ACP), mimicking the mineralizing pattern of the oriented assembly of ACP guided by amelogenin in the biomineralization of enamel. METHODS: CMC/ACP nanocomplex solution was first synthesized through the successive addition of carboxymethyl chitosan, calcium chloride, and dipotassium phosphate into distilled water. ACP nanoparticles were degraded by 1% NaClO from CMC/ACP nanocomplexes. The morphology of the particles at different periods was tested by transmission electron microscopy (TEM). The chimaeric peptides were added to guide the arrangement of ACP nanoparticles and to bind ACP nanoparticles to the demineralized enamel surface specifically. X-ray diffraction (XRD)/scanning electron microscope (SEM)/confocal laser scanning microscopy (CLSM)/nano-indentation tests were applied to check the remineralization effects. RESULTS: CMC/ACP nanocomplexes were obtained and could be kept without precipitation for a long time. After the degradation of NaClO and guidance of chimaeric peptides, ACP nanoparticles were arranged into oriented arrays before transforming into crystals, and the enamel-like crystals were tightly bound onto the demineralized surface. The newly formed enamel-like crystals were nearly well-organized and equipped with strong mechanical properties.

Modification of Titanium Substrates with Chimeric Peptides Comprising Antimicrobial and Titanium-Binding Motifs Connected by Linkers To Inhibit Biofilm Formation.

Bacterial adhesion and biofilm formation are the primary causes of implant-associated infection, which is difficult to eliminate and may induce failure in dental implants. Chimeric peptides with both binding and antimicrobial motifs may provide a promising alternative to inhibit biofilm formation on titanium surfaces. In this study, chimeric peptides were designed by connecting an antimicrobial motif (JH8194: KRLFRRWQWRMKKY) with a binding motif (minTBP-1: RKLPDA) directly or via flexible/rigid linkers to modify Ti surfaces. We evaluated the binding behavior of peptides using quartz crystal microbalance (QCM) and atomic force microscopy (AFM) techniques and investigated the effect of the modification of titanium surfaces with these peptides on the bioactivity of Streptococcus gordonii (S. gordonii) and Streptococcus sanguis (S. sanguis). Compared with the flexible linker (GGGGS), the rigid linker (PAPAP) significantly increased the adsorption of the chimeric peptide on titanium surfaces (p < 0.05). Concentration-dependent adsorption is consistent with a single Langmuir model, whereas time-dependent adsorption is in line with a two-domain Langmuir model. Additionally, the chimeric peptide with the rigid linker exhibited more effective antimicrobial ability than the peptide with the flexible linker. This finding was ascribed to the ability of the rigid linker to separate functional domains and reduce their interference to the maximum extent. Consequently, the performance of chimeric peptides with specific titanium-binding motifs and antimicrobial motifs against bacteria can be optimized by the proper selection of linkers. This rational design of chimeric peptides provides a promising alternative to inhibit the formation of biofilms on titanium surfaces with the potential to prevent peri-implantitis and peri-implant mucositis.