Postoperatively Noninvasive Optogenetic Stimulation via Upconversion Nanoparticles Enhancing Sciatic Nerve Repair.

The efficacy of electrical stimulation facilitating peripheral nerve regeneration is evidenced extensively, while the associated secondary damage resulting from repeated electrode invasion and indiscriminate stimulation is inevitable. Here, we present an optogenetics strategy that utilizes upconversion nanoparticles (UCNPs) to convert deeply penetrating near-infrared excitation into blue emission, which activates an adeno-associated virus-encoding ChR2 photoresponsive ion channel on cell membranes. The induced Ca2+ flux, similar to the ion flux in the electrical stimulation approach, efficiently regulates viability and proliferation, secretion of nerve growth factor, and neural function of RSC96 cells. Furthermore, deep near-infrared excitation is harnessed to stimulate autologous Schwann cells in situ via a UCNP-composited scaffold, which enhances nerve sprouting and myelination, consequently promoting functional recovery, electrophysiological restoration, and reinnervation of damaged nerves. This developed postoperatively noninvasive optogenetics strategy presents a novel, minimally traumatic, and enduring therapeutic stimulus to effectively promote peripheral nerve repair.

Integrated GelMA and interleukin 8-loaded liposome composite scaffold for endogenous BMSCs recruitment in bone repair.

Bone repair strategies, based on endogenous stem cell recruitment, can effectively avoid immune rejection and the low utilization of exogenous stem cells. Endogenous stem cells can be recruited to the implantation site by loading chemokines onto bone tissue-engineered scaffolds. However, challenges such as unstable chemokine activity and easy inactivation after implantation remain significant. In the present study, composite fiber scaffolds ((IL8@LIP)-GelMA) consisting of Interleukin 8 (IL8) -loaded liposomes and GelMA were constructed by electrospinning and photocrosslinking, and its ability to recruit bone marrow-derived mesenchymal stem cells (BMSCs) and immunomodulatory effect was investigated. Compared to GelMA loaded directly with IL8, scaffolds of (IL8@LIP)-GelMA demonstrated superior protection of IL8 activity, ensuring a slow and continuous release. Both in vivo and in vitro experiments demonstrated that the (IL8@LIP)-GelMA scaffolds effectively recruited BMSCs to the desired sites. Additionally, the (IL8@LIP)-GelMA scaffolds exhibited the capacity to recruit more macrophages to the implantation site. Importantly, they promoted the polarization of macrophages toward the M2 anti-inflammatory phenotype, facilitating the transition from the inflammatory stage to the tissue repair stage. Therefore, (IL8@LIP)-GelMA scaffolds show great potential for cell-free tissue engineering applications and provide insights into the loading mode of growth factors in scaffolds.

Conformal sulfidation of HKUST-1 for constructing porous Cu2S/CuO octahedrons realizing highly sensitive non-enzymatic glucose detection.

Metal-organic frameworks (MOFs) are believed to be promising precursors for constructing novel and efficient catalysts for glucose sensing. Herein, HKUST-1 precursors are first fabricated using a one-pot hydrothermal approach, and then HKUST-1 is converted into porous Cu2S/CuO octahedrons through conformal sulfidation with the help of OH-ions. The as-obtained Cu2S/CuO composite can provide rich electrochemical active sites and promoted electric transfer kinetics. Benefiting from these combined merits, the as-fabricated Cu2S/CuO composite is confirmed to be a high-performance catalyst, with high sensitivities of 8269.45 and 4140.82µA mM-1cm-2in the corresponding ranges of 0.05 ∼ 0.6 mM and 0.6 ∼ 1.2 mM, respectively. Moreover, the as-prepared electrode materials possess good anti-interference ability, reproducibility and long-term stability. This work opens up new avenues for the design and preparation of transition metal sulfide composites.

Ratiometric fluorescence detection of dopamine based on copper nanoclusters and carbon dots.

Nanoclusters for fluorescence detection are generally comprised of rare and expensive noble metals, and the nanoclusters based on more affordable transition metal have attracted increasing attention. This study designed a ratiometric fluorescent probe to detect dopamine (DA), an important neurotransmitter. With carbon dots encapsulated within silica (CDs@SiO2) as the reference, the emitted reference signal was almost unchanged due to the protection of inert silicon shell. Meanwhile, copper nanoclusters modified with 3-aminophenyl boronic acid (APBA-GSH-CuNCs) provided the sensing signal, in which the phenylboric acid could specifically recognize the cis-diol structure of DA, and caused the fluorescence quenching by photoinduced electron transfer. This dual emission ratiometric fluorescent probe exhibited high sensitivity and anti-interference, and was able to selectively responded to DA with a linear range of 0-1.4 mM, the detection limit of 5.6 nM, and the sensitivity of 815 mM-1. Furthermore, the probe successfully detected DA in human serum samples, yielding recoveries ranging from 92.5% to 102.7%. Overall, this study highlights the promising potential of this ratiometric probe for detecting DA.

Dendritic Oligoethylenimine Decorated Liposome with Augmented Corneal Retention and Permeation for Efficient Topical Delivery of Antiglaucoma Drugs.

The topically administered glaucoma medications usually encounter serious precorneal drug loss and low corneal penetration, leading to a low bioavailability. In addition, due to the complexity of glaucoma etiology, a single medication is often insufficient. In this work, we report a novel dendritic oligoethylenimine decorated liposome for codelivery of two antiglaucoma drugs, latanoprost and timolol. The liposome showed a uniform nanoscopic particle size, positive surface charge, and excellent dual-drug loading capacity. A prolonged precorneal retention is observed by using this liposomal delivery system. This liposomal delivery system presents increased cellular uptake and tight junctions opening capacity, contributing respectively to the transcellular and paracellular permeation, thereby enhancing the trans-corneal transportation. Following topical administration of one eye drop in brown Norway rats, the dual-drug-loaded liposome formulation resulted in a sustained and effective intraocular pressure reduction as long as 5 days, without inducing ocular inflammation, discomfort, and tissue damage.

Quasi-aligned Cu2S/Cu(OH)2nanorod arrays anchored on Cu foam as self-supported electrode for non-enzymatic glucose detection.

Self-supported Cu2S/Cu(OH)2composite nanorods for highly sensitive non-enzymatic glucose sensing werein situgrown on Cu foam by simple hydrothermal treatment of aligned Cu(OH)2nanorods. The physicochemical and electrochemical properties of the as-fabricated Cu2S/Cu(OH)2composite nanorods were characterized by scanning electron microscopy, transmission electron microscopy, x-ray diffraction, Raman spectroscope, x-ray photoelectron spectroscope, cyclic voltammetry, electrochemical impedance spectroscopy, amperometrici-tmeasurements. The mechanism of the composite nanorods produced on conductive substrates was also explored. The electrode exhibits a sensitivity of 9626.88µA mM-1cm-2towards glucose with good anti-interference ability, indicating it a promising electrode material for the enhanced non-enzymatic glucose detection.

Facile synthesis of Cu/Co-ZIF nanoarrays for non-enzymatic glucose detection.

Cu/Co-ZIF nanoflake arrays on carbon cloth are fabricated by controlling the introducing of Cu2+ions during the growth of Co-ZIF. The Cu/Co-ZIF-20 electrode prepared with 20 mM Cu2+possesses large electrochemically active surface area and bimetallic active sites, which can be revealed by cyclic voltammetry tests. The amperometrici-tmeasurements demonstrate that the Cu/Co-ZIF-20 electrode displays a wide linear range from 0.05 mM to 6.0 mM, and a high sensitivity of 1.03 mA mM-1cm-2. Good selectivity, repeatability and practical applicability indicate its promising application in enzyme-free glucose sensing.

[From Passive Application to Active Design -Restructuring and Reshaping of Core Specialty Knowledge Systems of Biomedical Engineering (Biomedical Materials Track) Undergraduate Education].

Biomedical engineering (BME) (biomedical materials track) is a typical field of interdisciplinary integration. Its specialty education simultaneously undertakes the duo reformation responsibilities for the new engineering education and the new medical education due to its unique strengths in interdisciplinary nature, comprehensive scope of knowledge, and status of being on the cutting edge of technology. We made an analysis, in this paper, of the opportunities and challenges faced by BME (biomedical materials track) specialty education on the basis of the trends and frontiers of development in biomedical materials in the world. From the perspective of new requirements raised by major national strategies and industrial development for the qualifications and competence of professionals specializing in biomedical materials, thorough reflections were made on the specialized education of BME (biomedical materials track) under the background of the new engineering education and the new medical education. Furthermore, we proposed herein to reconstruct the specialized core knowledge system according to the main line of the reactions and the responses between the biomedical materials and human bodies at different levels and set up a series of courses of biomedical materials science centered on Materiobiology as the core. We also proposed to establish a diversified integrated reform model of the training system incorporating production, learning, research and application for highly competent BME (biomedical materials track) professionals. This paper attempts to contribute to the solution of the major issue of how to train the innovative talents and leaders who will pioneer a new round of diagnosis and treatment technology revolution and the development of the medical device industry.

Hierarchically porous CuO spindle-like nanosheets grown on a carbon cloth for sensitive non-enzymatic glucose sensoring.

Herein, porous CuO spindle-like nanosheets were fabricated on a carbon cloth using a facile hydrothermal method, and surface morphology, microstructure, and glucose sensing performance were studied. The porous spindle-like nanosheets are constructed by nanoparticles and slit-like pores, exhibiting a hierarchical structure. When used for non-enzymatic glucose sensoring, the obtained CuO nanosheet electrode exhibits a wide linear range from 0.05 to 3.30 mM, a high sensitivity of 785.2 µA mM-1 cm-2 and a low detection limit of 0.22 µM (S/N = 3). Besides, good selectivity, stability, and reproducibility for glucose detection indicate a promising application of CuO nanosheet electrodes as non-enzymatic glucose sensors.

Co(OH)2 nanosheets decorated Cu(OH)2 nanorods for highly sensitive nonenzymatic detection of glucose.

Co(OH)2 nanosheets/Cu(OH)2 nanorods composite electrodes for non-enzymatic glucose detection were fabricated by electrodepositing Co(OH)2 nanosheets on Cu(OH)2 nanorods substrate grown directly on the copper sheet via a simple one-step reaction. The Co(OH)2 nanosheets/Cu(OH)2 nanorods composite electrode was characterized by scanning electron microscopy, energy dispersive x-ray spectroscopy, x-ray diffraction and x-ray photoelectron spectroscopy. The glucose sensing performance of the composite electrode was investigated by cyclic voltammetry and chronoamperometry. The composite electrode shows high sensitivity of 2366 µA mM-1 cm-2 up to 2 mM with a lower detection limit of 0.17 mM (S/N = 3). The composite electrode is highly selective to glucose in the presence of various substances that always co-exist with glucose in real blood samples. The response of the composite towards human blood serum was in good agreement with that of commercially available glucose sensors, suggesting that a promising electrode material for highly sensitive and selective non enzymatic detection of glucose can be envisioned.

Two-stage CNNs for computerized BI-RADS categorization in breast ultrasound images.

BACKGROUND: Quantizing the Breast Imaging Reporting and Data System (BI-RADS) criteria into different categories with the single ultrasound modality has always been a challenge. To achieve this, we proposed a two-stage grading system to automatically evaluate breast tumors from ultrasound images into five categories based on convolutional neural networks (CNNs). METHODS: This new developed automatic grading system was consisted of two stages, including the tumor identification and the tumor grading. The constructed network for tumor identification, denoted as ROI-CNN, can identify the region contained the tumor from the original breast ultrasound images. The following tumor categorization network, denoted as G-CNN, can generate effective features for differentiating the identified regions of interest (ROIs) into five categories: Category "3", Category "4A", Category "4B", Category "4C", and Category "5". Particularly, to promote the predictions identified by the ROI-CNN better tailor to the tumor, refinement procedure based on Level-set was leveraged as a joint between the stage and grading stage. RESULTS: We tested the proposed two-stage grading system against 2238 cases with breast tumors in ultrasound images. With the accuracy as an indicator, our automatic computerized evaluation for grading breast tumors exhibited a performance comparable to that of subjective categories determined by physicians. Experimental results show that our two-stage framework can achieve the accuracy of 0.998 on Category "3", 0.940 on Category "4A", 0.734 on Category "4B", 0.922 on Category "4C", and 0.876 on Category "5". CONCLUSION: The proposed scheme can extract effective features from the breast ultrasound images for the final classification of breast tumors by decoupling the identification features and classification features with different CNNs. Besides, the proposed scheme can extend the diagnosing of breast tumors in ultrasound images to five sub-categories according to BI-RADS rather than merely distinguishing the breast tumor malignant from benign.

Biomimetic Mineralization of Magnetic Iron Oxide Nanoparticles Mediated by Bi-Functional Copolypeptides.

Magnetite (Fe3O4) nanoparticles are widely used in multiple biomedical applications due to their magnetic properties depending on the size, shape and organization of the crystals. However, the crystal growth and morphology of Fe3O4 nanoparticles remain difficult to control without using organic solvent or a high temperature. Inspired by the natural biomineralization process, a 14-mer bi-functional copolypeptide, leveraging the affinity of binding Fe3O4 together with targeting ovarian cancer cell A2780, was used as a template in the biomimetic mineralization of magnetite. Alongside this, a ginger extract was applied as an antioxidant and a size-conditioning agent of Fe3O4 crystals. As a result of the cooperative effects of the peptide and the ginger extract, the size and dispersibility of Fe3O4 were controlled based on the interaction of the amino acid and the ginger extract. Our study also demonstrated that the obtained particles with superparamagnetism could selectively be taken up by A2780 cells. In summary, the Fe3O4-QY-G nanoparticles may have potential applications in targeting tumor therapy or angiography.

Impacts of Blast-Induced Traumatic Brain Injury on Expressions of Hepatic Cytochrome P450 1A2, 2B1, 2D1, and 3A2 in Rats.

The hepatic cytochrome P450 (CYP450) enzyme superfamily is one of the most important drug-metabolizing enzyme systems, which is responsible for the metabolism of a large number of clinically relevant medications used in traumatic brain injury (TBI) therapy. Modification of CYP450 expression may have important influences on drug metabolism and lead to untoward effects on those with narrow therapeutic windows. However, the impact of blast-induced TBI (bTBI) on the expression of CYP450 has received little attention. The subfamilies of CYP1A, 2B, 2D, and 3A account for about 85 % of all human drug metabolism of clinical significance. Therefore, in this study, we investigated the expressions of hepatic CYP1A2, CYP2B1, CYP2D1, and CYP3A2 in rats suffering bTBI. Meanwhile, we also measured some important cytokines in serum after injury, and calculated the correlation between these cytokines and the expressions of CYP1A2, CYP2B1, CYP2D1, and CYP3A2. The results showed that bTBI could significantly reduce mRNA expressions of CYP1A2, CYP2D1, and CYP3A2 at the early stage and induce the expressions from 48 h to 1 week after injury. The protein expressions of these CYP450s had all been downregulated from 24 to 48 h post- injury, and then began to elevate at 48 h after bTBI. The cytokines, IL-1ß, IL-2, IL-6, and TNF-α, increased significantly in the early phase, and began to reduce at the delayed phase of bTBI. The serum levels of IL-1ß, IL-6, and TNF-α but not IL-2 were significantly negative correlated with the mRNA expressions of CYP2B1 and CYP2D1 and the proteins expressions of CYP1A2, CYP2B1, CYP2D1, and CYP3A2. In conclusion, our work has, for the first time, indicated that bTBI has significant impact on the expressions of CYP1A2, CYP2B1, CYP2D1, and CYP3A2, which may be related to the cytokines induced by the injury.

One-pot synthesis of γ-MnS/reduced graphene oxide with enhanced performance for aqueous asymmetric supercapacitors.

In this work, γ-MnS/reduced graphene oxide composites (γ-MnS/rGO) were prepared using a facile one-pot hydrothermal method. As an electrode material for supercapacitors, the γ-MnS/rGO-60 composite obtained under dosages of graphene oxide  was 60 mg and exhibited an enhanced specific capacitance of 547.6 F g-1 at a current density of 1 A g-1, and outstanding rate capability (65% capacitance retention at 20 A g-1), with superior cycling stability and electrochemical reversibility. An asymmetric supercapacitor assembled from γ-MnS/rGO-60 composite and rGO (γ-MnS/rGO-60//rGO) showed a voltage window of 0-1.6 V and delivered a high energy density of 23.1 W h kg-1 at a power density of 798.8 W kg-1, and 15.9 W h kg-1 at 4.5 kW kg-1. Moreover, two such 1.0 × 1.0 cm2 devices connected together in series easily light up a group of LED lights, showing its potential practical application as an attractive energy storage device.

Fabrication and Protein Conjugation of Aligned Polypyrrole-Poly(L-lactic acid) Fibers Film with the Conductivity and Stability.

The conducting composite scaffold, including fiber-cores of aligned poly(L-lactic acid) (PLLA) and shell-layer of polypyrrole (PPy), was fabricated, and then bovine serum albumin (BSA) was conjugated on the PPy shell-layer. Aligned PLLA fibers (about 300 nm diameter) were obtained by electrospinning and rotating drum collection, and then coated by PPy nanoparticles (NPs, about 50 nm diameter) via chemical oxidation. The surface resistivity of PPy-PLLA fibers film were 0.971, 0.874 kΩ. cm at the fiber's vertical and parallel directions, respectively. The results of PPy-PLLA fibers film immersed in phosphate buffer saline for 8 d indicated that the fibers morphology and the film conductivity were not significantly changed, and the fluorescent images showed that FITC-labeled BSA (FITC-BSA) were successfully conjugated in the fibers film with carbodiimide chemistry, and the largest amount of FITC-BSA conjugated in the fibers film from 100 µg/mL proteins solution was 31.31 µg/cm2 due to lots of poly(glutamic acid) in surface-nanogrooves of the fibers surface. Under electrical stimulation of 100 mV, the fibers film was accompanied the release of all conjugated FITC-BSA with the detachment of some PPy NPs. These results suggested that PPy-PLLA fibers film would be potentially applied in the construction of degradable tissue engineering scaffold with protein factors, especially neurotrophic factors for nerve tissue repair.

Preparation of Polypyrrole-Protein Composite Films and the Electrochemically Controlled Release of Proteins.

It is fabricated that an electrically controlled release system based on the (poly-L-lactic acid)-mixed polypyrrole (PLLA-PPy) films through casting film of PPy and PLLA mixed solution on the glass plate, in which polyglutamic acid (PGlu)-doped PPy nanoparticles (NPs) with -50 nm diameter are synthesized via chemical oxidation. Surface conductivity of the composite film is (3.33 ± 2.01) x 10(-3) S/cm. Bovine serum albumin (BSA), as a model protein drug, is chemically linked onto the composite film via carbodiimide chemistry due to the good surface nano-structure of PLLA-PPy film. The releases of BSA from PLLA-PPy film under constant current and constant voltage can be achieved using the two-electrode electrochemical system. 6 h accumulative releases of BSA are 276 µg/cm2 and 176 µg/cm2 under 3 mA and 1 V electrical stimulation, respectively, accompanied with de-doping of PGlu and separation of a part of PPy NPs from the composite film. The results of cell experiment indicate that PGlu-doped PPy NPs in the prepared composite film have good cyto-compatibility. These results suggest that PPy-PLLA composite film would be able to be applied in the construction of degradable protein-drug-loaded scaffold for nerve tissue repair.

Tumor-targeted paclitaxel-loaded folate conjugated poly(ethylene glycol)-poly(L-lactide) microparticles produced by supercritical fluid technology.

The new biodegradable diblock copolymers poly(ethylene glycol)-poly(L-lactide) (PEG-PLLA) were synthesized and were chemically conjugated with folate (FA) in the PEG terminal ends to form FA-PEG-PLLA. Then the hydrophobic drug paclitaxel (PTX) loaded microparticles (PTX/FA-PEG-PLLA) were produced via solution enhanced dispersion by supercritical fluids (SEDS). These microparticles exhibited sphere-like shape by scanning electron microscopy observation and showed narrow hydrodynamic size distributions by dynamic light scattering measurement. Drug loading of PTX loaded microparticles was about 7-9% and the encapsulation efficiency of PTX loaded microparticles was about 18-23%. Flow cytometry and confocal laser scanning microscope analyses revealed that fluorescein isothiocyanate labeled FA conjugated microparticles presented significantly higher cellular uptake than FA-free group due to the FA-receptor-mediated endocytosis. In vitro cytotoxicity evaluation indicated that FA-PEG-PLLA expressed negligible cytotoxicity to mouse fibroblasts L929 cells. Moreover, PTX/FA-PEG-PLLA microparticles exhibited much higher anti-cancer efficacy than PTX/PEG-PLLA microparticles against human ovarian cancer SKOV3 cells. Nude mice xenografted with SKOV3 cells were used in biodistribution studies, the results indicated that an increased amount of PTX was accumulated in the tumor tissue deal with PTX/FA-PEG-PLLA microparticles. These results collectively suggested that PTX/FA-PEG-PLLA microparticles prepared by SEDS would have potential in anti-tumor applications as a tumor-targeted drug delivery formulation.

Cell adhesion and invasion inhibitory effect of an ovarian cancer targeting peptide selected via phage display in vivo.

Organ-specific metastasis is of great importance since most of the cancer deaths are caused by spread of the primary cancer to distant sites. Therefore, targeted anti-metastases therapies are needed to prevent cancer cells from metastasizing to different organs. The phage clone pc3-1 displaying peptide WSGPGVWGASVK selected by phage display had been identified which have high binding efficiency and remarkable cell specificity to SK-OV-3 cells. In the present work, the effects of selected cell-binding phage and cognate peptide on the cell adhesion and invasion of targeted cells were investigated. Results showed that the adhesive ability of SK-OV-3 to extracellular matrix was inhibited by pc3-1 and peptide WSGPGVWGASVK, and pc3-1 blocked SK-OV-3 cells attachment more effective than the cognate peptide. The peptide WSGPGVWGASVK suppressed the cell number of SK-OV-3 that attached to HUVECs monolayer up to 24% and could block the spreading of the attaching cells. Forthermore, the cognate peptide could inhibit the invasion of SK-OV-3 significantly. The number of invaded SK-OV-3 cells and invaded SK-OV-3-activated HUVECs pretreated with peptide WSGPGVWGASVK was decreased by 24.3% and 36.6%, respectively. All these results suggested that peptide WSGPGVWGASVK might possess anti-metastasis against SK-OV-3 cells.

Effects of combinations of BMP-2 with FGF-2 and/or VEGF on HUVECs angiogenesis in vitro and CAM angiogenesis in vivo.

Angiogenesis, a complex biologic process, is regulated by a large number of angiogenic factors, including vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). Whether Bone morphogenetic proteins-2 (BMP-2), the osteoinductive factor, could significantly reinforce the effect of VEGF and FGF-2 on angiogenesis has not been studied in detail. To study the positive effects of multiple growth factors on angiogenesis, HUVECs were treated with BMP-2, VEGF, or FGF-2 singly and in binary and ternary combinations. This study further investigates the optimal timing of the ternary combination of BMP-2, VEGF and FGF-2 for angiogenesis in the chorioallantoic membrane (FGF-2 CAM). Results of single applications of BMP-2, VEGF, or FGF-2 suggested that HUVECs angiogenesis could be promoted in a dose-dependent manner and that the optimal concentration of BMP, VEGF and FGF-2 was 10, 50 and 1 ng/mL, respectively. These results indicated that the angiogenic activity of VEGF and FGF-2 was amplified by combining with BMP-2. The ternary combination of BMP-2, VEGF and FGF-2 exhibited a positive and synergistic effect on HUVECs angiogenesis, with the lower concentrations of each factor (1 ng/mL of BMP-2, 25 ng/mL of VEGF and 0.1 ng/mL of FGF-2) being sufficient to show synergistic promotion. When VEGF and FGF-2 were added in the initial activation stage and BMP-2 was added in the maturation stage, both HUVECs angiogenesis in vitro and CAM angiogenesis in vivo could be enhanced more effectively. These results could provide a basis for the controlled release systems capable of delivering multiple factors sequentially to promote angiogenesis in tissue engineering.

Synergistic and sequential effects of BMP-2, bFGF and VEGF on osteogenic differentiation of rat osteoblasts.

In the present study, the effects of bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) on regulation of rat osteoblast (ROB) maturation in vitro were investigated. It was found that the proliferation, differentiation and mineralization of ROBs were all dose-dependently increased at particular times in the case of treatment with only one growth factor. To investigate the effects of combined treatment, ROBs were treated with either a single application of a relatively high dose of each growth factor, or binary/triple combined applications of relatively low doses of the growth factors. Osteogenic differentiation was significantly promoted in the triple combination treatment of BMP-2, VEGF and bFGF compared with the single or binary combination treatments. The optimal timing of the triple combination to enhance osteogenesis was also tested. When bFGF and VEGF were added in the early stage, and BMP-2 and VEGF were added in the late stage, osteogenic differentiation of ROBs could be enhanced more effectively. These results could be used to construct bone tissue engineering scaffolds that release growth factors sequentially.