Hydroxyapatite microbeads containing BMP-2 and quercetin fabricated via electrostatic spraying to encourage bone regeneration.

BACKGROUND: Hydroxyapatite (HAp) possesses osteoconductive properties, and its granular form can serve as an effective drug delivery vehicle for bone regeneration. Quercetin (Qct), a plant-derived bioflavonoid, is known to promote bone regeneration; however, its comparative and synergistic effects with the commonly used bone morphogenetic protein-2 (BMP-2) have not been investigated. METHODS: We examined the characteristics of newly formed HAp microbeads using an electrostatic spraying method and analyzed the in vitro release pattern and osteogenic potential of ceramic granules containing Qct, BMP-2, and both. In addition, HAp microbeads were transplanted into a rat critical-sized calvarial defect and the osteogenic capacity was assessed in vivo. RESULTS: The manufactured beads had a microscale size of less than 200 µm, a narrow size distribution, and a rough surface. The alkaline phosphatase (ALP) activity of osteoblast-like cells cultured with the BMP-2-and-Qct-loaded HAp was significantly higher than that of either Qct- or BMP-2-loaded HAp groups. The mRNA levels of osteogenic marker genes such as ALP and runt-related transcription factor 2 were found to be upregulated in the HAp/BMP-2/Qct group compared to the other groups. In micro-computed tomographic analysis, the amount of newly formed bone and bone surface area within the defect was significantly higher in the HAp/BMP-2/Qct group, followed by the HAp/BMP-2 and HAp/Qct groups, which is consistent with the histomorphometrical results. CONCLUSIONS: These results imply that electrostatic spraying can be an efficient strategy to produce homogenous ceramic granules and that the BMP-2-and-Qct-loaded HAp microbeads can serve as effective implants for bone defect healing.

Superior Alignment of Human iPSC-Osteoblasts Associated with Focal Adhesion Formation Stimulated by Oriented Collagen Scaffold.

Human-induced pluripotent stem cells (hiPSCs) can be applied in patient-specific cell therapy to regenerate lost tissue or organ function. Anisotropic control of the structural organization in the newly generated bone matrix is pivotal for functional reconstruction during bone tissue regeneration. Recently, we revealed that hiPSC-derived osteoblasts (hiPSC-Obs) exhibit preferential alignment and organize in highly ordered bone matrices along a bone-mimetic collagen scaffold, indicating their critical role in regulating the unidirectional cellular arrangement, as well as the structural organization of regenerated bone tissue. However, it remains unclear how hiPSCs exhibit the cell properties required for oriented tissue construction. The present study aimed to characterize the properties of hiPSCs-Obs and those of their focal adhesions (FAs), which mediate the structural relationship between cells and the matrix. Our in vitro anisotropic cell culture system revealed the superior adhesion behavior of hiPSC-Obs, which exhibited accelerated cell proliferation and better cell alignment along the collagen axis compared to normal human osteoblasts. Notably, the oriented collagen scaffold stimulated FA formation along the scaffold collagen orientation. This is the first report of the superior cell adhesion behavior of hiPSC-Obs associated with the promotion of FA assembly along an anisotropic scaffold. These findings suggest a promising role for hiPSCs in enabling anisotropic bone microstructural regeneration.

Evaluation of Mesenchymal Stem Cell Sheets Overexpressing BMP-7 in Canine Critical-Sized Bone Defects.

The aim of this study was to investigate the in vitro osteogenic capacity of bone morphogenetic protein 7 (BMP-7) overexpressing adipose-derived (Ad-) mesenchymal stem cells (MSCs) sheets (BMP-7-CS). In addition, BMP-7-CS were transplanted into critical-sized bone defects and osteogenesis was assessed. BMP-7 gene expressing lentivirus particles were transduced into Ad-MSCs. BMP-7, at the mRNA and protein level, was up-regulated in BMP-7-MSCs compared to expression in Ad-MSCs. Osteogenic and vascular-related gene expressions were up-regulated in BMP-7-CS compared to Ad-MSCs and Ad-MSC sheets. In a segmental bone-defect model, newly formed bone and neovascularization were enhanced with BMP-7-CS, or with a combination of BMP-7-CS and demineralized bone matrix (DBM), compared to those in control groups. These results demonstrate that lentiviral-mediated gene transfer of BMP-7 into Ad-MSCs allows for stable BMP-7 production. BMP-7-CS displayed higher osteogenic capacity than Ad-MSCs and Ad-MSC sheets. In addition, BMP-7-CS combined with demineralized bone matrix (DBM) stimulated new bone and blood vessel formation in a canine critical-sized bone defect. The BMP-7-CS not only provides BMP-7 producing MSCs but also produce osteogenic and vascular trophic factors. Thus, BMP-7-CS and DBM have therapeutic potential for the treatment of critical-sized bone defects and could be used to further enhance clinical outcomes during bone-defect treatment.

142Development of an alginate-gelatin bioink enhancing osteogenic differentiation by gelatin release.

Hydrogels are natural bioink options for cellular printing due to their high-water content and permeable three-dimensional (3D) polymeric structure, which are favorable for cellular anchoring and metabolic activities. To increase the functionality of hydrogels as bioinks, biomimetic components are often incorporated, such as proteins, peptides, and growth factors. In this study, we aimed to enhance the osteogenic activity of a hydrogel formulation by integrating both the release and retention of gelatin so that gelatin serves as both an indirect support for released ink component on cells nearby and a direct support for encapsulated cells inside a printed hydrogel, thereby fulfills two functions. Methacrylate-modified alginate (MA-alginate) was chosen as the matrix because it has a low cell adhesion effect due to the absence of ligands. The gelatin-containing MA-alginate hydrogel was fabricated, and gelatin was found to remain in the hydrogel for up to 21 days. The gelatin remaining in the hydrogel had positive effects on encapsulated cells, especially on cell proliferation and osteogenic differentiation. The gelatin released from the hydrogel affected the external cells, showing more favorable osteogenic behavior than the control sample. It was also found that the MA-alginate/gelatin hydrogel could be used as a bioink for printing with high cell viability. Therefore, we anticipate that the alginate-based bioink developed in this study could potentially be used to induce osteogenesis in bone tissue regeneration.

Support-less ceramic 3D printing of bioceramic structures using a hydrogel bath.

Volumetric bone tissue defects are beyond the intrinsic regenerative capacity of bone tissue. With the recent development of ceramic 3D printing, various bioceramic scaffolds that can induce bone regeneration are being actively developed. However, hierarchical bone is complex, with overhanging structures that require additional sacrificial support during ceramic 3D printing. Not only can this increase the overall process time and material consumption, but breaks and cracks may occur when sacrificial supports are removed from fabricated ceramic structures. In this study, a support-less ceramic printing (SLCP) process using a hydrogel bath was developed to facilitate the manufacture of complex bone substitutes. A hydrogel bath, consisting of pluronic P123 with temperature-sensitive properties, mechanically supported the fabricated structure when the bioceramic ink was extruded into the bath and promoted the cement reaction to cure the bioceramic. SLCP enables the fabrication of complex bone constructs with overhanging structures, such as the mandible and maxillofacial bones, with reduced overall processing time and material consumption. Scaffolds fabricated by SLCP showed more cell adhesion, higher cell growth rate, and osteogenic protein expression due to their rougher surface than conventionally printed scaffolds. Hybrid scaffolds were fabricated by SLCP to co-print cells and bioceramics, and SLCP provided a cell-friendly environment, exhibiting high cell viability. SLCP enables control of the shape of various cells, bioactive substances, and bioceramics and thus can be used as an innovative 3D bioprinting technique to manufacture complex hierarchical bone structures.

Multifunctional bone substitute using carbon dot and 3D printed calcium-deficient hydroxyapatite scaffolds for osteoclast inhibition and fluorescence imaging.

Multifunctional bone substitute materials (BSM) have gained considerable attention with the exponential increase in aging populations. The development of hybrid materials for diagnosis and therapy of bone-related diseases and dysfunctions, especially, has been a significant challenge in the biological and the biomedical field, due to the shortage of agents with specificity and selectivity toward bone. In this study, a hybrid material, referred as Alen-CDs@CDHA, fabricated from alendronate-conjugated carbon dots (Alen-CDs) and calcium-deficient hydroxyapatite (CDHA, the mineral component of bones) scaffolds is offered as a novel multifunctional BSM for in vivo osteoclasts deactivation and fluorescence imaging. The fluorescent Alen-CDs were hydrothermally prepared using phytic acid as carbon source, followed by conjugating alendronate, for controlled alendronate release and fluorescent imaging under acidic conditions. As-prepared fluorescent Alen-CDs were consecutively immobilized on surfaces of CDHA scaffolds, exhibiting high affinity by bisphosphonate group, easily fabricated from α-tricalcium phosphate (α-TCP) paste using three-dimensional (3D) printing system. The resultant Alen-CDs@CDHA caused a significant decrease (> 50%) in viability of osteoclasts at 7 days after in vitro treatment. Furthermore, when Alen-CDs@CDHA was implanted in balb/c nude mice for in vivo evaluation, we found Alen-CDs@CDHA to be suitable for bone imaging through fluorescence signals, without necrosis or inflammatory symptoms in the epidermal tissues. Thus, these observations offer new opportunities for a novel and revolutionary use of Alen-CDs@CDHA as highly specific multifunctional BSM for bone diagnosis and imaging, and as bone-specific drug delivery materials, eventually providing anti-osteoclastogenic treatments solution for degenerative bone disorders. STATEMENT OF SIGNIFICANCE: Alen-CDs@CDHA significantly reduced the viability of osteoclasts and fluorescently imaged in vivo after transplantation, releasing drug via pH modulation. The development of fluorescence materials for bone imaging remains still a major challenge in the biomedical field owing to the shortage of selectivity and specificity. The results could lead to improvements in bone treatment strategies, as it could reduce the invasiveness of procedures and the associated negative outcomes, and increase the precision of strategies. Further, we believe that this study will be of interest to the readership of your journal as clearly focuses on the advancement of a biomaterial, where we have engineered a substance to substitute bone and integrate with a living system.

Signal-Decay Based Approach for Visualization of Buried Defects in 3-D Printed Ceramic Components Imaged with Help of Optical Coherence Tomography.

We propose the use of Optical Coherence Tomography (OCT) as a tool for the quality control of 3-D-printed ceramics. Test samples with premeditated defects, namely single- and two-component samples of zirconia, titania, and titanium suboxides, were printed by stereolithography-based DLP (Digital Light Processing) processes. The OCT tomograms obtained on the green samples showed the capability of the method to visualize variations in the layered structure of the samples as well as the presence of cracks and inclusions at depths up to 130 µm, as validated by SEM images. The structural information was visible in cross-sectional images as well as in plan-view images. The optical signal measured from the printed zirconia oxide and titanium oxide samples showed strong attenuation with depth and could be fit with an exponential decay curve. The variations of the decay parameter correlated very well with the presence of defects and material variation. When used as an imaging quantity, the decay parameter projects the position of the defects into 2-D (X,Y) coordinates. This procedure can be used in real time, it reduces the data volume up to 1000 times, and allows for faster subsequent data analysis and transfer. Tomograms were also obtained on sintered samples. The results showed that the method can detect changes in the optical properties of the green ceramics caused by sintering. Specifically, the zirconium oxide samples became more transparent to the light used, whereas the titanium suboxide samples became entirely opaque. In addition, the optical response of the sintered zirconium oxide showed variations within the imaged volume, indicating material density variations. The results presented in this study show that OCT provides sufficient structural information on 3-D-printed ceramics and can be used as an in-line tool for quality control.

Fabrication of color-graded feldspathic dental prosthetics for aesthetic and restorative dentistry.

OBJECTIVE: Feasibility investigation of natural teeth shades replication on dental prosthetics fabricated via functionally graded additive manufacturing (FGAM) using combination of feldspathic porcelain (FP) and yttrium aluminum garnet cerium (Y3Al5O12:Ce, YAG:Ce) as a promising esthetic restoration option. METHODS: Color-graded feldspathic crown fabrication parameter through FGAM method was comprehensively examined from the slurry rheology, cure depth, debinding to sintering temperature. Effect of light absorbent also checked towards overcuring reaction during UV exposure by the shape comparison. Lastly, the flexural bending strength measured following ISO 6872:2015 to assure the applicability. Applying the studied parameter, natural teeth shades then imitated and investigated by alteration of FP and FP + 0.1 wt% YAG:Ce (Y-FP). Generated color across the structure captured through mobile camera, interpreted through the CIELAB coordinate and the gradation confirmed by the color differences (ΔE00) calculated using CIEDE2000 formula. RESULT: Parameter study indicated that 70 wt% of FP slurry with 3 wt% dispersant and 0.2 wt% light absorbent is favored. It produces excellent flowability in our FGAM system with less overcuring justified by edge margin reduction from 95.65° to 90.00° after UV exposure on rectangle shapes masking. The obtain structure also offers adequate flexural bending strength of 106.26 MPa (FP) and 101.36 MPa (Y-FP) after sintering at 780 °C. This validated the materials as class 2 dental prosthetics citing ISO 6872:2015. Color gradation was verified by the yellow b* value reduction (14.8 to -3.33) as it shifted from cervical to incisal area while ΔE00 further affirmed the differences from each segment in comparison with the FP and Y-FP. SIGNIFICANCE: Color gradation was successfully replicated by FP and YAG:Ce composition shift via FGAM technique. This result highlights the potential of FGAM as an alternative for fabricating dental prosthetics with high efficiency and improved esthetic appeal.

Enhancement of properties of a cell-laden GelMA hydrogel-based bioink via calcium phosphate phase transition.

To improve the properties of the hydrogel-based bioinks, a calcium phosphate phase transition was applied, and the products were examined. We successfully enhanced the mechanical properties of the hydrogels by adding small amounts (< 0.5 wt%) of alpha-tricalcium phosphate (α-TCP) to photo-crosslinkable gelatin methacrylate (GelMA). As a result of the hydrolyzing calcium phosphate phase transition involvingα-TCP, which proceeded for 36 h in the cell culture medium, calcium-deficient hydroxyapatite was produced. Approximately 18 times the compressive modulus was achieved for GelMA with 0.5 wt%α-TCP (20.96 kPa) compared with pure GelMA (1.18 kPa). Although cell proliferation decreased during the early stages of cultivation, both osteogenic differentiation and mineralization activities increased dramatically when the calcium phosphate phase transition was performed with 0.25 wt%α-TCP. The addition ofα-TCP improved the printability and fidelity of GelMA, as well as the structural stability and compressive modulus (approximately six times higher) after three weeks of culturing. Therefore, we anticipate that the application of calcium phosphate phase transition to hydrogels may have the potential for hard tissue regeneration.

Fabrication of multifunctional alginate microspheres containing hydroxyapatite powder for simultaneous cell and drug delivery.

Novel alginate-hydroxyapatite hybrid microspheres were developed for simultaneous delivery of drugs and cells as a multifunctional bone substitute for osteoporotic bone tissue regeneration. The microspheres were used to enhance osteogenesis and to carry and deliver quercetin, a representative phytoestrogen that controls bone tissue regeneration metabolism in osteoporosis patients, through sustained release over a long period. To overcome quercetin's hydrophobicity and low solubility in aqueous environments, we added it to the surface of hydroxyapatite (HAp) nanoparticles before mixing them with an alginate solution. The homogeneous distribution of the HAp nanoparticles in the alginate solution was essential for preventing nozzle clogging and achieving successfully fabricated hybrid microspheres. To this end, a 3D ultrasonic treatment was applied. Electrostatic microencapsulation was then used to fabricate hybrid alginate-HAp microspheres containing quercetin and cells. The microspheres were approximately 290.7 ± 42.5 µm (aspect ratio of 1). The sustained release of quercetin was confirmed during a test period of 20 weeks. The cells in the hybrid microspheres maintained good cell viability during the entire testing period, and their osteogenic differentiation behavior was boosted by the presence of HAp. Thus, osteogenic differentiation could be greatly improved by adding quercetin. These novel multi-biofunctional hybrid microspheres have great potential for the regeneration of osteoporotic bone tissue at indeterminate defect sites.

Greater Plasma Protein Adsorption on Mesoporous Silica Nanoparticles Aggravates Atopic Dermatitis.

Purpose: The protein corona surrounding nanoparticles has attracted considerable attention as it induces subsequent inflammatory responses. Although mesoporous silica nanoparticles (MSN) are commonly used in medicines, cosmetics, and packaging, the inflammatory effects of the MSN protein corona on the cutaneous system have not been investigated till date. Methods: We examined the greater plasma protein adsorption on MSN leads to serious inflammatory reactions in Dermatophagoides farinae extract (DFE)-induced mouse atopic dermatitis (AD)-like skin inflammation because of increased uptake by keratinocytes. Results: We compare the AD lesions induced by MSN and colloidal (non-porous) silica nanoparticles (CSN), which exhibit different pore architectures but similar dimensions and surface chemistry. MSN-corona treatment of severe skin inflammation in a DFE-induced in vivo AD model greatly increases mouse ear epidermal thickness and infiltration of immune cells compared with the CSN-corona treatment. Moreover, MSN-corona significantly increase AD-specific immunoglobulins, serum histamine, and Th1/Th2/Th17 cytokines in the ear and lymph nodes. MSN-corona induce more severe cutaneous inflammation than CSN by significantly decreasing claudin-1 expression. Conclusion: This study demonstrates the novel impact of the MSN protein corona in inducing inflammatory responses through claudin-1 downregulation and suggests useful clinical guidelines for MSN application in cosmetics and drug delivery systems.

Multifunctional Calcium-Deficient Hydroxyl Apatite-Alginate Core-Shell-Structured Bone Substitutes as Cell and Drug Delivery Vehicles for Bone Tissue Regeneration.

In this work, we fabricated unique coiled-structured bioceramics contained in hydrogel beads for simultaneous drug and cell delivery using a combination of bone cement chemistry and bioprinting and characterized them. The core of the calcium-deficient hydroxyl apatite (CDHA) contains quercetin, which is a representative phytoestrogen isolated from onions and apples, to control the metabolism of bone tissue regeneration through sustained release over a long period of time. The shell consists of an alginate hydrogel that includes preosteoblast MC3T3-E1 cells. Ceramic paste and hydrogel were simultaneously extruded to fabricate core-shell beads through the inner and outer nozzles, respectively, of a concentric nozzle system based on a material-extruding-based three-dimensional (3D) printing system. The formation of beads and the coiled ceramic core is related to both alginate concentration and printing conditions. The size of the microbeads and the thickness of the coiled structure could be controlled by adjusting the nozzle conditions. The whole process was carried out at physiological conditions (37 °C) to be gentle on the cells. The alginate shell undergoes solidification by cross-linking in CaCl2 or monocalcium phosphate monohydrate (MCPM) solution, while the hardening and cementation of the α-tricalcium phosphate (α-TCP) core to CDHA are subsequently initiated by immersion in phosphate-buffered saline solution. This process replaces the typical sintering of ceramic processing to prevent damage to the hydrogel, cells, and drugs in the beads. The cell-loaded beads were then cultured in cell culture media where the cells could maintain good viability during the entire testing period, which was over 50 days. Cell growth and elongation were observed even in the alginate along the CDHA coiled structure over time. Sustained release of quercetin without any initial burst was also confirmed during a test period of 120 days. These novel structured microbeads with multibiofunctionality can be used as new bone substitutes for hard tissue regeneration in indeterminate defect sites.

3D Bioprinting of In Vitro Models Using Hydrogel-Based Bioinks.

Coronavirus disease 2019 (COVID-19), which has recently emerged as a global pandemic, has caused a serious economic crisis due to the social disconnection and physical distancing in human society. To rapidly respond to the emergence of new diseases, a reliable in vitro model needs to be established expeditiously for the identification of appropriate therapeutic agents. Such models can be of great help in validating the pathological behavior of pathogens and therapeutic agents. Recently, in vitro models representing human organs and tissues and biological functions have been developed based on high-precision 3D bioprinting. In this paper, we delineate an in-depth assessment of the recently developed 3D bioprinting technology and bioinks. In particular, we discuss the latest achievements and future aspects of the use of 3D bioprinting for in vitro modeling.

Selective Detection of an Infection Biomarker by an Osteo-Friend Scaffold: Development of a Multifunctional Artificial Bone Substitute.

Developments in three-dimensional (3D) printing technologies have led to many potential applications in various biomedical fields, especially artificial bone substitutes (ABSs). However, due to the characteristics of artificial materials, biocompatibility and infection remain issues. Here, multifunctional ABSs have been designed to overcome these issues by the inclusion of a biochemical modality that allows simultaneous detection of an infection biomarker by osteo-friend 3D scaffolds. The developed multifunctional scaffolds consist of calcium-deficient hydroxyapatite (CDHA), which has a similar geometric structure and chemical composition to human bone, and gold nanoparticles (Au NPs), which assists osteogenesis and modulates the fluorescence of labels in their microenvironment. The Au NPs were subsequently conjugated with fluorescent dye-labeled probe DNA, which allowed selective interaction with a specific target biomarker, and the fluorescent signal of the dye was temporally quenched by the Au NP-derived Förster resonance energy transfer (FRET). When the probe DNA unfolded to bind to the target biomarker, the fluorescence signal was recovered due to the increased distance between the dye and Au NPs. To demonstrate this sensing mechanism, a microbial oligonucleotide was selected as a target biomarker. Consequently, the multifunctional scaffold simultaneously facilitated osteogenic proliferation and the detection of the infection biomarker.

Enhanced healing of rat calvarial defects with 3D printed calcium-deficient hydroxyapatite/collagen/bone morphogenetic protein 2 scaffolds.

In this paper, we mainly to evaluate the newly formed bone using the Calcium deficient hydroxyapatite (CDHA)/collagen-based bio-ceramic scaffold as Bone Morphogenetic Protein-2 (BMP-2) carrier in rat calvarial critical-sized bone defect. In the real-time PCR analysis, the CDHA/collagen scaffold loaded rhBMP-2 group showed significantly enhanced results of bone-related gene expression (p < 0.05). In the in vivo study, the micro-CT showed that the main bone formation parameters of percent bone volume and trabecular number of the two experiment groups (CDHA/Collagen (CDHA) group, BV/TV: 14.21 ± 3.20, Tb.N: 2.37 ± 0.50; CDHA/Collagen/rhBMP-2(BMP) group, BV/TV: 14.51 ± 3.12, Tb.N: 2.75 ± 0.65) were significantly higher than those of the control (Blank, BV/TV: 3.25 ± 1.25, Tb.N: 0.57 ± 0.20) group (p < 0.05). Although there was no significant difference between the two experimental groups, the BMP group results were slightly higher than those of the CDHA group (p > 0.05). Moreover, the histological results also supported the micro-CT results. The scaffold of CDHA/collagen seems to be a suitable bio-ceramic carrier loaded rhBMP-2, and appears to enhance new bone formation and bone regeneration in bone defect after implantation.

Bone-targeting carbon dots: effect of nitrogen-doping on binding affinity.

Novel fluorescent carbon dots (CDs) for bone imaging were fabricated via a facile hydrothermal method using alendronate in the absence of a nitrogen-doping precursor to enhance bone affinity. One-step synthesized alendronate-based CDs (Alen-CDs) had strong binding activity for calcium-deficient hydroxyapatite (CDHA, the mineral component of bones) scaffold, rat femur, and bone structures of live zebrafish. This was attributed to the bisphosphonate group present on the CD surface, even after carbonization. For comparison, the surface effects of nitrogen-doped CDs obtained using ethylenediamine (EDA), i.e., Alen-EDA-CDs, were also investigated, focusing on the targeting ability of distinct surface functional groups when compared with Alen-CDs. An in vivo study to assess the impact on bone affinity revealed that Alen-CDs effectively accumulated in the bone structures of live zebrafish larvae after microinjections, as well as in the bone tissues of femur extracted from rats. Moreover, Alen-CD-treated zebrafish larvae had superior toleration, retaining skeletal fluorescence for 7 days post-injection (dpi). The sustainable capability, surpassing that of Alizarin Red S, suggests that Alen-CDs have the potential for targeted drug delivery to damaged bone tissues and provides motivation for additional in vivo investigations. To our knowledge, this is the first in vitro, ex vivo, and in vivo demonstration of direct bone-targeted deliveries, supporting the use of fluorescent CDs in the treatment of various bone diseases such as osteoporosis, Paget's disease, and metastatic bone cancer.

Construction of human induced pluripotent stem cell-derived oriented bone matrix microstructure by using in vitro engineered anisotropic culture model.

Bone tissue has anisotropic microstructure based on collagen/biological apatite orientation, which plays essential roles in the mechanical and biological functions of bone. However, obtaining an appropriate anisotropic microstructure during the bone regeneration process remains a great challenging. A powerful strategy for the control of both differentiation and structural development of newly-formed bone is required in bone tissue engineering, in order to realize functional bone tissue regeneration. In this study, we developed a novel anisotropic culture model by combining human induced pluripotent stem cells (hiPSCs) and artificially-controlled oriented collagen scaffold. The oriented collagen scaffold allowed hiPSCs-derived osteoblast alignment and further construction of anisotropic bone matrix which mimics the bone tissue microstructure. To the best of our knowledge, this is the first report showing the construction of bone mimetic anisotropic bone matrix microstructure from hiPSCs. Moreover, we demonstrated for the first time that the hiPSCs-derived osteoblasts possess a high level of intact functionality to regulate cell alignment. © 2017 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 360-369, 2018.

Fast and sensitive near-infrared fluorescent probes for ALP detection and 3d printed calcium phosphate scaffold imaging in vivo.

Alkaline phosphatase (ALP) is a critical biological marker for osteoblast activity during early osteoblast differentiation, but few biologically compatible methods are available for its detection. Here, we describe the discovery of highly sensitive and rapidly responsive novel near-infrared (NIR) fluorescent probes (NIR-Phos-1, NIR-Phos-2) for the fluorescent detection of ALP. ALP cleaves the phosphate group from the NIR skeleton and substantially alters its photophysical properties, therefore generating a large "turn-on" fluorescent signal resulted from the catalytic hydrolysis on fluorogenic moiety. Our assay quantified ALP activity from 0 to 1.0UmL-1 with a 10-5-10-3UmL-1 limit of detection (LOD), showing a response rate completed within 1.5min. A potentially powerful approach to probe ALP activity in biological systems demonstrated real-time monitoring using both concentration- and time-dependent variations of endogenous ALP in live cells and animals. Based on high binding affinity to bone tissue of phosphate moiety, bone-like scaffold-based ALP detection in vivo was accessed using NIR probe-labeled three-dimensional (3D) calcium deficient hydroxyapatite (CDHA) scaffolds. They were subcutaneously implanted into mice and monitored ALP signal changes using a confocal imaging system. Our results suggest the possibility of early-stage ALP detection during neo-bone formation inside a bone defect, by in vivo fluorescent evaluation using 3D CDHA scaffolds.

Magnesium phosphate ceramics incorporating a novel indene compound promote osteoblast differentiation in vitro and bone regeneration in vivo.

Incorporating bioactive molecules into synthetic ceramic scaffolds is challenging. In this study, to enhance bone regeneration, a magnesium phosphate (MgP) ceramic scaffold was incorporated with a novel indene compound, KR-34893. KR-34893 induced the deposition of minerals and expression of osteoblast marker genes in primary human bone marrow mesenchymal stem cells (BMSCs) and a mouse osteoblastic MC3T3-E1 cell line. Analysis of the mode of action showed that KR-34893 induced the phosphorylation of MAPK/extracellular signal-regulated kinase and extracellular signal-regulated kinase, and subsequently the expression of bone morphogenetic protein 7, accompanied by SMAD1/5/8 phosphorylation. Accordingly, KR-34893 was incorporated into an MgP scaffold prepared by 3D printing at room temperature, followed by cement reaction. KR-34893-incorporated MgP (KR-MgP) induced the expression of osteoblast differentiation marker genes in vitro. In a rat calvaria defect model, KR-MgP scaffolds enhanced bone regeneration and increased bone volume compared with MgP scaffolds, as assessed by micro-computed tomography and histological analyses. In conclusion, we developed a method for producing osteoinductive MgP scaffolds incorporating a bioactive organic compound, without high temperature sintering. The KR-MgP scaffolds enhanced osteoblast activation in vitro and bone regeneration in vivo.

Design and preparation of bioactive glasses with hierarchical pore networks.

Hierarchically giant-, macro-, and meso-porous 3D bioactive glass scaffolds with good bone-forming bioactivity in vitro were synthesized by using a combination of sol-gel, double polymers templating, and rapid prototyping techniques.