Critical size bone defects are regularly treated by auto-and allograft transplantation. However, such treatments require to harvest bone from patient donor sites, with often limited tissue availability or risk of donor site morbidity. Not requiring bone donation, three-dimensionally (3D) printed implants and biomaterial-based tissue engineering (TE) strategies promise to be the next generation therapies for bone regeneration. We present here polylactic acid (PLA)-bioactive glass (BG) composite scaffolds manufactured by fused deposition modeling (FDM), involving the fabrication of PLA-BG composite filaments which are used to 3D print controlled open-porous and osteoinductive scaffolds. We demonstrated the printability of PLA-BG filaments as well as the bioactivity and cytocompatibility of PLA-BG scaffolds using pre-osteoblast MC3T3E1 cells. Gene expression analyses indicated the beneficial impact of BG inclusions in FDM scaffolds regarding osteoinduction, as BG inclusions lead to increased osteogenic differentiation of human adipose-derived stem cells in comparison to pristine PLA. Our findings confirm that FDM is a convenient additive manufacturing technology to develop PLA-BG composite scaffolds suitable for bone tissue engineering.

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45S5 Bioglass® (BG) scaffolds with high porosity (>90%) were coated with genipin cross-linked gelatin (GCG) and further incorporated with poly(p-xylyleneguanidine) hydrochloride (PPXG). The obtained GCG coated scaffolds maintained the high porosity and well interconnected pore structure. A 26-fold higher compressive strength was provided to 45S5 BG scaffolds by GCG coating, which slightly retarded but did not inhibit the in vitro bioactivity of 45S5 BG scaffolds in SBF. Moreover, the scaffolds were made antibacterial against both Gram-positive and Gram-negative bacteria by using polyguanidine, i.e. PPXG, in this study. Osteoblast-like cells (MG-63) were seeded onto PPXG and GCG coated scaffolds. PPXG was biocompatible with MG-63 cells at a low concentration (10 μg mL−1). MG-63 cells were shown to attach and spread on both uncoated and GCG coated scaffolds, and the mitochondrial activity measurement indicated that GCG coating had no negative influence on the cell proliferation behavior of MG-63 cells. The developed novel antibacterial bioactive 45S5 BG-based composite scaffolds with improved mechanical properties are promising candidates for bone tissue engineering

Bioactive glasses (BAGs) are being investigated for the repair and reconstruction of bone defects, as they exhibit osteoconductive and osteostimulatory potential. However, successful bone regeneration requires also the neovascularization of the construct which is, among other factors, guided by vascular endothelial growth factor (VEGF). In this study, BAG S53P4 (53% SiO2 , 23% Na2 O, 20% CaO, 4% P2 O5 ) is investigated in relation to VEGF-release and response of fibroblast cells. Human CD-18CO fibroblasts were cultivated in contact with different granules of different sizes (0.5-0.8 mm, 1.0-2.0 mm, and 2.0-3.15 mm) and at different concentrations (0-1 wt/vol % of BAG) for 72 h. The analysis of morphology revealed no toxic effect for all granule sizes and concentrations. Compared with the reference, lactate dehydrogenase-activity of CCD-18CO cells increased in contact with BAG samples. The VEGF release from CCD-18CO fibroblasts cultured on different granule sizes and at different concentrations after 72 h of incubation was quantified. It was found that particles of 0.5-0.8 mm and 1.0-2.0 mm in size enhanced VEGF release, whereas BAG particle sizes of 2.0-3.15 mm led to inhibition of VEGF release. The results are relevant to understand the influence of the particle size and concentration of BAG S53P4 on VEGF expression and neovascularization.

The influence of carbonate substitution (4.4 wt%, mixed A/B type) in hydroxyapatite ceramics for bone remodeling scaffolds was investigated by separately analyzing the response of pre-osteoblasts and osteoclast-like cells. Carbonated hydroxyapatite (CHA) (Ca(PO)(CO)(OH)(CO)-CHA), mimicking the chemical composition of natural bone mineral, and pure hydroxyapatite (HA) (Ca(PO)(OH)-HA) porous ceramics were processed to obtain a similar microstructure and surface physico-chemical properties (grain size, porosity ratio and pore size, surface roughness and zeta potential). The biological behavior was studied using MC3T3-E1 pre-osteoblastic and RAW 264.7 monocyte/macrophage cell lines. Chemical dissolution in the culture media and resorption lacunae produced by osteoclasts occur with both HA and CHA ceramics, but CHA exhibits much higher dissolution and greater bioresorption ability. CHA ceramics promoted a significantly higher level of pre-osteoblast proliferation. Osteoblastic differentiation, assessed by qRT-PCR of RUNX2 and COLIA2, and pre-osteoclastic proliferation and differentiation were not significantly different on CHA or HA ceramics but cell viability and metabolism were significantly greater on CHA ceramics. Thus, the activity of both osteoclast-like and osteoblastic cells was influenced by the carbonate substitution in the apatite structure. Furthermore, CHA showed a particularly interesting balance between biodegradation, by osteoclasts and chemical dissolution, and osteogenesis through osteoblasts' activity, to stimulate bone regeneration. It is hypothesized that this amount of 4.4 wt% carbonate substitution leads to an adapted concentration of calcium in the fluid surrounding the ceramic to stimulate the activity of cells. These results highlight the superior biological behavior of microporous 4.4 wt% A/B CHA ceramics that could beneficially replace the commonly used HA of biphasic calcium phosphates for future applications in bone tissue engineering.

Highly porous 45S5 bioactive glass-based scaffolds prepared by foam replication method were coated with cellulose nanowhiskers by dip coating method. The obtained cellulose nanowhisker-coated scaffolds retained the high porosity and interconnected pore structure. The cellulose coating improved the mechanical properties of the scaffolds and did not hinder their bioactivity in simulated body fluid. In vitro biocompatibility assessment was carried out by qualitative evaluation of the morphology of osteoblast-like cells (MG-63) seeded onto the scaffolds. The cells were shown to attach and spread on both uncoated scaffolds and cellulose nanowhisker-coated scaffolds, thus cellulose nanowhisker coating seems to have no negative influence on the behavior of MG-63 cells. The obtained bioactive and biocompatible composite scaffolds represent promising candidates for bone tissue engineering applications

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