A High-Throughput Platform to Identify Small-Molecule Inhibitors of CRISPR-Cas9.

The precise control of CRISPR-Cas9 activity is required for a number of genome engineering technologies. Here, we report a generalizable platform that provided the first synthetic small-molecule inhibitors of Streptococcus pyogenes Cas9 (SpCas9) that weigh <500 Da and are cell permeable, reversible, and stable under physiological conditions. We developed a suite of high-throughput assays for SpCas9 functions, including a primary screening assay for SpCas9 binding to the protospacer adjacent motif, and used these assays to screen a structurally diverse collection of natural-product-like small molecules to ultimately identify compounds that disrupt the SpCas9-DNA interaction. Using these synthetic anti-CRISPR small molecules, we demonstrated dose and temporal control of SpCas9 and catalytically impaired SpCas9 technologies, including transcription activation, and identified a pharmacophore for SpCas9 inhibition using structure-activity relationships. These studies establish a platform for rapidly identifying synthetic, miniature, cell-permeable, and reversible inhibitors against both SpCas9 and next-generation CRISPR-associated nucleases.

Phosphorylation-Inducing Chimeric Small Molecules.

Small molecules have been classically developed to inhibit enzyme activity; however, new classes of small molecules that endow new functions to enzymes via proximity-mediated effect are emerging. Phosphorylation (native or neo) of any given protein-of-interest can alter its structure and function, and we hypothesized that such modifications can be accomplished by small molecules that bring a kinase in proximity to the protein-of-interest. Herein, we describe phosphorylation-inducing chimeric small molecules (PHICS), which enable two example kinases-AMPK and PKC-to phosphorylate target proteins that are not otherwise substrates for these kinases. PHICS are formed by linking small-molecule binders of the kinase and the target protein, and exhibit several features of a bifunctional molecule, including the hook-effect, turnover, isoform specificity, dose and temporal control of phosphorylation, and activity dependent on proximity (i.e., linker length). Using PHICS, we were able to induce native and neo-phosphorylations of BRD4 by AMPK or PKC. Furthermore, PHICS induced a signaling-relevant phosphorylation of the target protein Bruton's tyrosine kinase in cells. We envision that PHICS-mediated native or neo-phosphorylations will find utility in basic research and medicine.

Synthesis of an Aminooxy Derivative of the GM3 Antigen and Its Application in Oxime Ligation.

The anomeric aminooxy GM3 trisaccharide cancer antigen (Neu5Acα2,3Galß1,4Glcß-ONH2) has been chemically synthesized using a linear glycosylation approach. The key step involves a highly α(2,3)-stereoselective sialylation to a galactose acceptor. The Neu5Acα2,3Gal intermediate was functionalized as a donor for a [2 + 1] glycosylation, including a glucose acceptor that featured an O-succinimidyl group on the reducing end as an aminooxy precursor. The fully deprotected anomeric aminooxy GM3 trisaccharide was then conjugated to the immunologically relevant zwitterionic polysaccharide PS A1 via an oxime link.

Enhanced Immune Response Against the Thomsen-Friedenreich Tumor Antigen Using a Bivalent Entirely Carbohydrate Conjugate.

The Thomsen-Friedenreich (TF) antigen is a key target for the development of anticancer vaccines, and this ongoing challenge remains relevant due to the poor immunogenicity of the TF antigen. To overcome this challenge, we adopted a bivalent conjugate design which introduced both the TF antigen and the Thomsen-nouveau (Tn) antigen onto the immunologically relevant polysaccharide A1 (PS A1). The immunological results in C57BL/6 mice revealed that the bivalent, Tn-TF-PS A1 conjugate increased the immune response towards the TF antigen as compared to the monovalent TF-PS A1. This phenomenon was first observed with enzyme-linked immunosorbent assay (ELISA) where the bivalent conjugate generated high titers of IgG antibodies where the monovalent conjugate generated an exclusive IgM response. Fluorescence-activated cell sorting (FACS) analysis also revealed increased binding events to the tumor cell lines MCF-7 and OVCAR-5, which are consistent with the enhanced tumor cell lysis observed in a complement dependent cytotoxicity (CDC) assay. The cytokine profile generated by the bivalent construct revealed increased pro-inflammatory cytokines IL-17 and IFN-γ. This increase in cytokine concentration was matched with an increase in cytokine producing cells as observed by ELISpot. We hypothesized the mechanisms for this phenomenon to involve the macrophage galactose N-acetylgalactosamine specific lectin 2 (MGL2). This hypothesis was supported by using biotinylated probes and recombinant MGL2 to measure carbohydrate-protein interactions.

Production of a mouse monoclonal IgM antibody that targets the carbohydrate Thomsen-nouveau cancer antigen resulting in in vivo and in vitro tumor killing.

The construction of a tumor-associated carbohydrate antigen-zwitterionic polysaccharide conjugate, Thomsen-nouveau-polysaccharide A1 (Tn-PS A1, where Tn = D-GalpNAc), has led to the development of a carbohydrate binding monoclonal antibody named Kt-IgM-8. Kt-IgM-8 was produced via hybridoma from Tn-PS A1 hyperimmunized Jackson Laboratory C57BL/6 mice, splenocytes and the murine myeloma cell line Sp2/0Ag14 with subsequent cloning on methyl cellulose semi-solid media. This in-house generated monoclonal antibody negates binding influenced from peptides, proteins, and lipids and preferentially binds monovalent Tn antigen as noted by ELISA, FACS, and glycan array technologies. Kt-IgM-8 demonstrated in vitro and in vivo tumor killing against the Michigan Cancer Foundation breast cell line 7 (MCF-7). In vitro tumor killing was observed using an LDH assay that measured antibody-induced complement-dependent cytotoxicity and these results were validated in an in vivo passive immunotherapy approach using an MCF-7 cell line-derived xenograft model. Kt-IgM-8 is effective in killing tumor cells at 30% cytotoxicity, and furthermore, it demonstrated approximately 40% reduction in tumor growth in the MCF-7 model.

Sialyl-Tn Polysaccharide A1 as an Entirely Carbohydrate Immunogen: Synthesis and Immunological Evaluation.

Sialyl Thomsen-nouveau (STn) is a tumor-associated carbohydrate antigen (TACA) that is overexpressed in a variety of carcinomas such as breast, ovarian, and colon cancer. In normal tissue, STn is not detectable, which is critical for opportunities in developing cancer immunotherapies. A novel, entirely carbohydrate, semisynthetic STn-polysaccharide (PS) A1 conjugate was prepared and evaluated in C57BL/6 mice. STn-PS A1 was combined with commercially available monophosphoryl lipid A-based adjuvant, and after immunization, ELISA indicated a strong immune response for inducing anti-STn IgM/IgG antibodies. The specificity of these antibodies was concomitantly investigated using FACS analysis, and the results indicated excellent cell surface binding events to STn-expressing cancer cell lines MCF-7 and OVCAR-5. An INF-γ ELISpot assay was conducted to further confirm a robust cellular immunity invoked by STn-PS A1. Most importantly, the raised antibodies conferred complement-dependent cellular cytotoxicity against MCF-7 and OVCAR-5 cells.

Immunological evaluation of the entirely carbohydrate-based Thomsen-Friedenreich - PS B conjugate.

PS B, a naturally occurring CD4(+) T-cell simulating zwitterionic polysaccharide from Bacteroides fragilis ATCC 25285/NCTC 9343, was conjugated with aminooxy Thomsen Friedenreich (TF or T) [α-d-Gal-(1,3)-ß-d-GalNAc-ONH2] tumor antigen. Immunization in Jax C57BL/6, followed by ELISA revealed IgM and IgG antibody TF specificity. FACS data noted preferential binding to TF-laced MCF-7 cells but not to HCT-116 cells.

Synthesis of the tumor associative α-aminooxy disaccharide of the TF antigen and its conjugation to a polysaccharide immune stimulant.

The α-aminooxy derivative of the Thomsen-Friedenriech tumor associated carbohydrate antigen has been synthesized in 11 steps utilizing a D-GalN3 acceptor carrying a pre-installed α-N-hydroxysuccinimidyl moiety. The natural α linkage was prepared in high selectivity employing a suitably protected D-GalN3-thioglycoside donor with N-hydroxysuccinimide. With access to α-TF-ONH2, the preparation of the TF-PS A1 vaccine candidate ensued smoothly through oxime bond formation.

Investigation of the Degradation of LiPF6- in Polar Solvents through Deep Potential Molecular Dynamics.

The nonaqueous electrolyte based on lithium hexafluorophosphate (LiPF6) is the dominant liquid electrolyte in lithium-ion batteries (LIBs). However, trace protic impurities, including H3O+, alcohols, and hydrofluoric acid (HF), can trigger a series of side reactions that lead to rapid capacity fading in high energy density LIBs. It is worth noting that this degradation process is highly dependent on the polarity of the solvents. In this work, a deep potential (DP) model is trained with a certain commercial electrolyte formula through a machine learning method. H3O+ is anchored with polar solvents, making it difficult to approach the PF6-, and suppressing the degradation process quickly at room temperature. Control experiments and simulations at different temperatures or concentrations are also performed to verify it. This work proposes a precise model to describe the solvation structure quantitatively and offers a new perspective on the degradation mechanism of PF6- in polar solvents.

Reliability of using generic flow conditions to quantify aneurysmal haemodynamics: A comparison against simulations incorporating boundary conditions measured in vivo.

BACKGROUND AND OBJECTIVES: Initiation, growth, and rupture of intracranial aneurysms are believed to be closely related to their local haemodynamic environment. While haemodynamics can be characterised by use of computational fluid dynamics (CFD), its reliability depends heavily upon accurate assumption of the boundary conditions. Herein, we compared the simulated aneurysmal haemodynamics obtained by use of generic boundary conditions against those obtained under flow conditions measured in vivo. METHODS: We prospectively recruited 19 patients with intracranial aneurysms requiring 3-dimensional rotational angiography, during which blood pressure at the internal carotid artery was probed by catheter and flowrate measured by a dedicated software tool. Using these flow conditions measured in vivo, we quantified the aneurysmal haemodynamics for each patient by CFD, and then compared the results with those derived from a generic condition reported in the literature, in terms of the time-averaged wall shear stress (TAWSS), oscillatory shear index (OSI), relative residence time (RRT), and percentage of the intra-aneurysmal flow (PIAF). In addition, the effects on aneurysmal haemodynamics of different outflow strategies (splitting method vs. Murray's Law) and simulation schemes (transient vs. steady-state) relative to each flow condition were also assessed. RESULTS: Differences in the simulated TAWSS (-6.08 ± 10.64 Pa, p = 0.001), OSI (0.06 ± 0.13, p = 0.001), and PIAF (-0.05 ± 0.20, p = 0.012) between the patient-specific and generic boundary conditions were found to be statistically significant, in contrast to that in the RRT (49 ± 307 Pa-1, p = 0.062). Outflow strategies did not yield statistically significant differences in any of the investigated parameters (all p > 0.05); rather, the resulting parameters were found to be in good correlations (all r > 0.71, p < 0.001). Difference between the aneurysmal TAWSS and the WSS derived from cycle-averaged flowrate condition was found to be minor (0.66 ± 1.36 Pa, p = 0.000), so was that between PIAFs obtained respectively from the transient and steady-state simulations (0.02 ± 0.05, p = 0.000). CONCLUSIONS: Incorporating into simulation the patient-specific boundary conditions is critical for CFD to characterise aneurysmal haemodynamics, while outflow strategies may not introduce significant uncertainties. Steady-state simulation incorporating the cycle-averaged flow condition may produce unbiased WSS and PIAF compared to the transient analysis.

Effect of Dual Pore Size Architecture on In Vitro Osteogenic Differentiation in Additively Manufactured Hierarchical Scaffolds.

The combination of macro- and microporosity is a potent manner of enhancing osteogenic potential, but the biological events leading to this increase in osteogenesis are not well understood. In this study, we investigated the effect of a dual pore size scaffold on the physical and biological properties, with the hypothesis that cell condensation is the determining factor for enhanced osteogenic differentiation. To this end, a hierarchical scaffold possessing a dual (large and small) pore size was fabricated by combining two additive manufacturing techniques: melt electrospinning writing (MEW) and fused deposition modeling (FDM). The scaffolds showed a mechanical stiffness of 23.2 ± 1.5 MPa similar to the FDM control scaffold, while the hybrid revealed an increased specific surface area of 1.4 ± 0.1 m2/g. The scaffold was cultured with primary human osteoblasts for 28 days, which showed enhanced cell adhesion and proliferation. The hierarchical structure was also beneficial for in vitro alkaline phosphate activity and mineralization and showed an increased expression of osteogenic protein and genes. Mesenchymal condensation markers related to osteoblastic differentiation (CDH2, RhoA, Rac1, and Cdc42) were upregulated in the hybrid construct, demonstrating that the MEW membrane provided an environment more suitable for the recapitulation of cell condensation, which in turn leads to higher osteogenic differentiation. In summary, this study demonstrated that the hierarchical scaffold developed in this paper leads to a significant improvement in the scaffold properties such as increased specific surface area, initial cell adhesion, cell proliferation, and in vitro osteogenesis.

Can pre-ozonation be combined with gravity-driven membrane filtration to treat shale gas wastewater?

Low-cost gravity-driven membrane (GDM) filtration has the potential to efficiently manage highly decentralized shale gas wastewater (SGW). In this work, the feasibility of combining low dosage pre-ozonation with the GDM process was evaluated in the treatment of SGW. The results showed that pre-ozonation significantly increased the stable flux (372%) of GDM filtration, while slightly deteriorating the quality of the effluent water in terms of organic content (-14%). These results were mainly attributed to the conversion of macromolecular organics to low-molecular weight fractions by pre-ozonation. Interestingly, pre-ozonation markedly increased the flux (198%) in the first month of operation also for a GDM process added with granular activated carbon (GGDM). Nevertheless, the flux of O3-GGDM systems dropped sharply around the 25th day of operation, which might be due to the rapid accumulation of pollutants in the high flux stage and the formation of a dense fouling layer. Pre-ozonation remarkably influenced the microbial community structure. And O3-GDM systems were characterized by distinct core microorganisms, which might degrade specific organics in SGW. Furthermore, O3-GDM outperformed simple GDM as a pretreatment for RO. These findings can provide valuable references for combining oxidation technologies with the GDM process in treating refractory wastewater.

Chemical synthesis and immunological evaluation of entirely carbohydrate conjugate Globo H-PS A1.

An anticancer, entirely carbohydrate conjugate, Globo H-polysaccharide A1 (Globo H-PS A1), was chemically prepared and immunologically evaluated in C57BL/6 mice. Tumor associated carbohydrate antigen Globo H hexasaccharide was synthesized in an overall 7.8% yield employing a convergent [3 + 3] strategy that revealed an anomeric aminooxy group used for conjugation to oxidized PS A1 via an oxime linkage. Globo H-PS A1, formulated with adjuvants monophosphoryl lipid A and TiterMax® Gold. After immunization an antigen specific immune response was observed in ELISA with anti-Globo H IgG/IgM antibodies. Specificity of the corresponding antibodies was determined by FACS showing cell surface binding to Globo H-positive cancer cell lines MCF-7 and OVCAR-5. The anti-Globo H antibodies also exhibited complement-dependent cellular cytotoxicity against MCF-7 and OVCAR-5 cells.

Stimulation of osteogenesis and angiogenesis by micro/nano hierarchical hydroxyapatite via macrophage immunomodulation.

Biomaterial topography-based strategies are regarded as an effective way to regulate the osteoimmune environment which plays an indispensable role in the bone regeneration process. The rapid development of manufacture techniques makes it possible to investigate the cell-topography interactions by preparing various micro and nano-topographical surfaces on biomaterials. Still, it is a challenge to prepare well-defined micro/nano hierarchical structures of bioceramics due to the inherent brittleness of ceramic materials. Also, the correlation between osteoimmunomodulation initiated by micro/nano hierarchical topographies and the tissue regeneration outcomes is unclear. In this study, we prepared well-defined micro/nano hierarchical structures on hydroxyapatite (HA) bioceramics through the combination of the photolithography and hydrothermal techniques. Three different microscale circular patterns (4 µm, 12 µm and 36 µm) and nanotopographies (nanoneedle, nanosheet and nanorod) were fabricated by changing the size of the mask and the condition of the hydrothermal reaction. The macrophage responses on the nanoneedle structures with different micropatterns were investigated and the micro/nano hierarchical structures with appropriate pattern sizes could either promote or alleviate the macrophage polarization, which further affected the outcomes of the osteogenic differentiation of human bone marrow stromal cells (hBMSCs) and angiogenic activity of human umbilical vein endothelial cells (HUVECs). Our study demonstrated that osteoimmunomodulation could be manipulated via tuning the micro/nano hierarchical structures, which could lead to a new strategy for the development of bone biomaterials with favorable osteoimmunomodulatory properties.

Recent Progress in the Construction of Functional Artificial Bone by Cytokine-Controlled Strategies.

OBJECTIVE: Combining artificial scaffolds with stimulatory factors to reconstruct lost bone tissues is one of the hottest research directions. The purpose of this review was to conduct a retrospective survey on the latest reports on artificial bone fabrication with functional cytokines. DATA SOURCES: The status of related scientific research from the year 2005 to 2018 was analyzed through the mode of literature retrieval in PubMed and VIP Database. The retrieval words are as follows: "bone tissue engineering," "angiogenesis," "cytokines," "osteogenesis," "biomimetic bone marrow," "sol-gel," "delivery system," and the corresponding Chinese words. STUDY SELECTION: After reading through the title and abstract for early screening, the full text of relevant studies was evaluated and those not related with this review had been ruled out. RESULTS: According to the literature retrospective survey, there were three key points for the successful construction of functional artificial bones: (1) the continuous supply of relatively low concentration of cytokines during the required period; (2) the delivery of two or more cytokines essential to the process and ensure the relatively spatial independence to reduce the unnecessary interference; and (3) supporting the early-stage angiogenesis and late-stage osteogenesis, respectively, regulating and balancing the crosslinking of both to avoid the surface ossification that would probably block the osteogenesis inside. CONCLUSIONS: The synergistic effect of both angiogenic factors and osteogenic factors applied in bone regeneration is a key point in the combined functional artificial bone. Through analysis, comparison, and summary of the current strategies, we proposed that the most promising one is to mimic the natural bone marrow function to facilitate the regeneration process and ensure the efficient repair of large weight-bearing bone defect.

Europium-doped mesoporous silica nanosphere as an immune-modulating osteogenesis/angiogenesis agent.

Although much research has gone into the design of nanomaterials, inflammatory response still impedes the capacity of nanomaterial-induced tissue regeneration. In-situ incorporation of nutrient elements in silica-based biomaterials has emerged as a new option to endow the nanomaterials modulating biological reactions. In this work, europium-doped mesoporous silica nanospheres (Eu-MSNs) were successfully synthesized via a one-pot method. The nanospheres (size of 280-300 nm) possess uniformly spherical morphology and mesoporous structure, and well distributed Eu elements. The nanospheres show distinct fluorescent property at 615 nm for potential bio-labeling. Noticeably, the Eu-MSNs stimulate pro-inflammatory response of macrophages and induce a modulated immune microenvironment, which further activates the osteogenic differentiation of bone marrow stromal cells (BMSCs) as well as angiogenic activity of human umbilical vein endothelial cells (HUVECs). During the process, osteogenesis-related genes (e.g. ALP, OCN, OPN and COL-I) of BMSCs, and angiogenesis-related genes (e.g. CD31, MMP9, VEGFR1/2, and PDGFRα/ß) of HUVECs were significantly upregulated by Eu-MSNs modulating immune environment of macrophages. The in vivo study further demonstrated that the Eu-MSNs could not only stimulate osteogenesis by accelerating the new bone formation at critical-sized cranial defect site, but also support the blood vessel formation as well as collagen deposition and re-epithelialization at chronic skin wound sites, showing an improved angiogenesis activity when comparing with MSNs alone. Given the easy handling characteristics and extensive application potential, the results suggest that Eu-MSNs could be used as immunity-modulated osteogenesis/angiogenesis agent for skin and bone regeneration.

Copper-doped mesoporous silica nanospheres, a promising immunomodulatory agent for inducing osteogenesis.

The application of mesoporous silica nanospheres (MSNs) loaded with drugs/growth factors to induce osteogenic differentiation of stem cells has been trialed by a number of researchers recently. However, limitations such as high cost, complex fabrication and unintended side effects from supraphysiological concentrations of the drugs/growth factors represent major obstacles to any potential clinical application in the near term. In this study we reported an in situ one-pot synthesis strategy of MSNs doped with hypoxia-inducing copper ions and systematically evaluated the nanospheres by in vitro biological assessments. The Cu-containing mesoporous silica nanospheres (Cu-MSNs) had uniform spherical morphology (∼100nm), ordered mesoporous channels (∼2nm) and homogeneous Cu distribution. Cu-MSNs demonstrated sustained release of both silicon (Si) and Cu ions and controlled degradability. The Cu-MSNs were phagocytized by immune cells and appeared to modulate a favorable immune environment by initiating proper pro-inflammatory cytokines, inducing osteogenic/angiogenic factors and suppressing osteoclastogenic factors by the immune cells. The immune microenvironment induced by the Cu-MSNs led to robust osteogenic differentiation of bone mesenchymal stem cells (BMSCs) via the activation of Oncostation M (OSM) pathway. These results suggest that the novel Cu-MSNs could be used as an immunomodulatory agent with osteostimulatory capacity for bone regeneration/therapy application. STATEMENT OF SIGNIFICANCE: In order to stimulate both osteogenesis and angiogenesis of stem cells for further bone regeneration, a new kind of hypoxia-inducing copper doped mesoporous silica nanospheres (Cu-MSNs) were prepared via one-pot synthesis. Biological assessments under immune environment which better reflect the in vivo response revealed that the nanospheres possessed osteostimulatory capacity and had potential as immunomodulatory agent for bone regeneration/therapy application. The strategy of introducing controllable amount of therapeutic ions instead of loading expensive drugs/growth factors in mesoporous silica nanosphere provides new options for bioactive nanomaterial functionalization.

3D printing of biomaterials with mussel-inspired nanostructures for tumor therapy and tissue regeneration.

Primary bone cancer brings patients great sufferings. To deal with the bone defects resulted from cancer surgery, biomaterials with good bone-forming ability are necessary to repair bone defects. Meanwhile, in order to prevent possible tumor recurrence, it is essential that the remaining tumor cells around bone defects are completely killed. However, there are few biomaterials with the ability of both cancer therapy and bone regeneration until now. Here, we fabricated a 3D-printed bioceramic scaffold with a uniformly self-assembled Ca-P/polydopamine nanolayer surface. Taking advantage of biocompatibility, biodegradability and the excellent photothermal effect of polydopamine, the bifunctional scaffolds with mussel-inspired nanostructures could be used as a satisfactory and controllable photothermal agent, which effectively induced tumor cell death in vitro, and significantly inhibited tumor growth in mice. In addition, owing to the nanostructured surface, the prepared polydopamine-modified bioceramic scaffolds could support the attachment and proliferation of rabbit bone mesenchymal stem cells (rBMSCs), and significantly promoted the formation of new bone tissues in rabbit bone defects even under photothermal treatment. Therefore, the mussel-inspired nanostructures in 3D-printed bioceramic exhibited a remarkable capability for both cancer therapy and bone regeneration, offering a promising strategy to construct bifunctional biomaterials which could be widely used for therapy of tumor-induced tissue defects.

Stimulation of osteogenesis and angiogenesis of hBMSCs by delivering Si ions and functional drug from mesoporous silica nanospheres.

Multifunctional bioactive materials with the ability to stimulate osteogenesis and angiogenesis of stem cells play an important role in the regeneration of bone defects. However, how to develop such biomaterials remains a significant challenge. In this study, we prepared mesoporous silica nanospheres (MSNs) with uniform sphere size (∼90 nm) and mesopores (∼2.7 nm), which could release silicon ions (Si) to stimulate the osteogenic differentiation of human bone marrow stromal cells (hBMSCs) via activating their ALP activity, bone-related gene and protein (OCN, RUNX2 and OPN) expression. Hypoxia-inducing therapeutic drug, dimethyloxaloylglycine (DMOG), was effectively loaded in the mesopores of MSNs (D-MSNs). The sustained release of DMOG from D-MSNs could stabilize HIF-1α and further stimulated the angiogenic differentiation of hBMSCs as indicated by the enhanced VEGF secretion and protein expression. Our study revealed that D-MSNs could combine the stimulatory effect on both osteogenic and angiogenic activity of hBMSCs. The potential mechanism of D-MSN-stimulated osteogenesis and angiogenesis was further elucidated by the supplementation of cell culture medium with pure Si ions and DMOG. Considering the easy handling characteristics of nanospheres, the prepared D-MSNs may be applied in the forms of injectable spheres for minimally invasive surgery, or MSNs/polymer composite scaffolds for bone defect repair. The concept of delivering both stimulatory ions and functional drugs may offer a new strategy to construct a multifunctional biomaterial system for bone tissue regeneration.

Mesoporous bioactive glass nanolayer-functionalized 3D-printed scaffolds for accelerating osteogenesis and angiogenesis.

The hierarchical microstructure, surface and interface of biomaterials are important factors influencing their bioactivity. Porous bioceramic scaffolds have been widely used for bone tissue engineering by optimizing their chemical composition and large-pore structure. However, the surface and interface of struts in bioceramic scaffolds are often ignored. The aim of this study is to incorporate hierarchical pores and bioactive components into the bioceramic scaffolds by constructing nanopores and bioactive elements on the struts of scaffolds and further improve their bone-forming activity. Mesoporous bioactive glass (MBG) modified ß-tricalcium phosphate (MBG-ß-TCP) scaffolds with a hierarchical pore structure and a functional strut surface (∼100 nm of MBG nanolayer) were successfully prepared via 3D printing and spin coating. The compressive strength and apatite-mineralization ability of MBG-ß-TCP scaffolds were significantly enhanced as compared to ß-TCP scaffolds without the MBG nanolayer. The attachment, viability, alkaline phosphatase (ALP) activity, osteogenic gene expression (Runx2, BMP2, OPN and Col I) and protein expression (OPN, Col I, VEGF, HIF-1α) of rabbit bone marrow stromal cells (rBMSCs) as well as the attachment, viability and angiogenic gene expression (VEGF and HIF-1α) of human umbilical vein endothelial cells (HUVECs) in MBG-ß-TCP scaffolds were significantly upregulated compared with conventional bioactive glass (BG)-modified ß-TCP (BG-ß-TCP) and pure ß-TCP scaffolds. Furthermore, MBG-ß-TCP scaffolds significantly enhanced the formation of new bone in vivo as compared to BG-ß-TCP and ß-TCP scaffolds. The results suggest that application of the MBG nanolayer to modify 3D-printed bioceramic scaffolds offers a new strategy to construct hierarchically porous scaffolds with significantly improved physicochemical and biological properties, such as mechanical properties, osteogenesis, angiogenesis and protein expression for bone tissue engineering applications, in which the incorporation of nanostructures and bioactive components into the scaffold struts synergistically play a key role in the improved bone formation.