This study was conducted to investigate the effect of magnetic nanoparticle composite scaffold under a pulsed electromagnetic field on bone marrow mesenchymal stem cells (BMSCs), which was achieved by examining the biological behaviors of cell adhesion, proliferation and differentiation on the surface of the scaffolds. This may provide some experimental evidence for the use of magnetic nanoparticles in medical application. The magnetic nanoparticle composite scaffolds were evaluated and characterized by the following indexes: the cell proliferation was detected by the CCK-8 method, the alkaline phosphatase (ALP) activity was examined by a detection kit, and the expression of type I collagen and osteocalcin gene were evaluated by RT-PCR. The CCK-8 test showed that there was no significant difference in Group A (BMSCs-seeded magnetic scaffolds under the electromagnetic field), B (BMSCs-seeded magnetic scaffolds) and C (BMSCs cultured alone) (P>0.05). The value for the ALP activity in Group A was higher than the other two groups. In addition, the RT-PCR results showed that the expression of type I collagen gene in Group A was enhanced (P<0.05), suggesting that the magnetic nanoparticles combined with the pulsed electromagnetic field had a positive effect on the osteogenic differentiation of BMSCs. However, the expression of osteocalcin was not significantly different in three groups (P>0.05). To conclude, magnetic nanoparticles may induce the osteogenic differentiation with the action of the pulsed electromagnetic field.

Objective Study prepared a novel two-phase magnetic nanocomposite scaffolds (PLGA/Col-I-PLGA/n-HA/ Fe2O3),through various biological performance testing,evaluation and to explore the feasibility of bone tissue engineering scaffolds.Methods Preparation of two-phase magnetic nanocomposite scaffolds (PLGA/Col-I-PLGA/n-HA/Fe2O3) by cryogenic rapidprototyping method,using electronic detection machine scaffold flexural,compressive strength,elastic modulus to evaluate the mechanical properties by electron microscopy ultrastructure scaffold;in the medium (ethanol) immersion assay scaffold porosity between the scaffold and bone marrow mesenchymal stem cells co-cultured detect its biocompatibility.Results Duplex magnetic nanocomposite scaffold mechanical test results show it has good mechanical properties,electron microscopy showed uniform pore size distribution of the upper and lower layers,the upper layer of cartilage with smaller pore size,good fusion between successive phase,pore size and porosity of the test results showed cartilage aperture layer scaffold to 189um,a porosity of 86.5％.Aperture bone scaffold layer is 364um,a porosity of 77.1％,in line with the double scaffold design requirements.Duplex between magnetic nano composite scaffold co-cultured with bone marrow mesenchymal stem cells,showed that bone marrow mesenchymal stem cell proliferation effect is very good,better able to promote the differentiation of cells for the purpose described biphasic magnetic nanocomposite scaffolds have good biocompatibility.Conclusion Duplex magnetic nanocomposite scaffolds (PLGA/Col-I-PLGA/n-HA/Fe2O3) has good mechanical properties and biocompatibility,pore size and pore growth rate of cell adhesion requirements,and normaljoint subchondral bone and cartilage physiological structure closer,is expected to be able to better repair osteoarthritis and other diseases caused by trauma or cartilage and subchondral bone damage under.

Objective To prepare porous magnetic nanocomposite artificial bone scaffolds (n-HA/PLLA/Fe2O3) using a low-temperature rapid prototyper, and to characterize the properties of the scaffold. Methods A low-tem-perature rapid prototyper was applied to fabricate the porous magnetic nanocomposite scaffolds with different mass ra-tios of n-HA/PLLA/Fe2O3. The medium (ethanol) immersion assay was used to examine the scaffold porosity. Mechani-cal tests, including bending, compression and elastic modulus, were performed to investigate the scaffold mechanical properties. A scanning electron microscope was used to observe the scaffold microstructures. Phosphate buffer solution (PBS) soaking was performed to study the scaffold degradation behavior by monitoring the changes of pH value and the scaffold mechanical properties. Results With the increase of the n-HA and Fe2O3 content, the tensile strength of scaf-fold gradually decreased, while the elastic modulus increased. The bending strength reached the maximum (111.9 MPa) when the mass fraction of n-HA and Fe2O3 was 10%, at which time the scaffold with a porosity of 90%showed a rough fracture surface. The scaffold had a porosity of 83%when the mass fraction of n-HA and Fe2O3 was 5%, with a large number of dimples being observed. If the mass fraction increased to 15%, a porosity of 80%was obtained and the fac-ture surface became relatively smooth. In the degradation study, the pH value of the medium decreased with time and the scaffold mechanical properties had certain attenuation. Conclusion The scaffold of 10%n-HA and Fe2O3 obtained bet-ter mechanical properties and degradation behavior, which would facilitate the selection of the porous magnetic nano-composite artificial bone scaffold with best performance.

BACKGROUND: As is known to all, cartilage tissue engineering has three elements, seed cells,scaffold materials, and cytokines. Scaffold materials play an important role in cartilage tissue engineering.OBJECTIVE: To search and review the literatures about tissue-engineered cartilage scaffold materials in the last decade,and to discuss problems and the development direction of the scaffolds.METHODS: A computer-based search of relevant articles published from 2006 to 2016 was conducted in PubMed and CNKI using the key words of "cartilage tissue engineering, natural scaffold materials, synthetic scaffold materials,composite scaffolds,nanometer materials" in English and Chinese, respectively.RESULTS AND CONCLUSION:Scaffold materials in cartilage tissue engineering have different sources and kinds.Natural materials, synthetic materials and nanometer materials have their own advantages and disadvantages. Any single material is difficult to meet the clinical requirements of the cartilage. Material compounds with different proportions can be used to prepare scaffolds with good mechanical properties, high porosity, good compatibility and degradation. But there are still some problems, such as excessive speed of scaffold degradation and excessive cell growth. The current research is in in vitro experimental stage mostly, and the application in clinic has not been enforced yet due to small size and lack of a long-term follow up.

Inorganic/organic hybrid scaffolds have great potential for tissue engineering applications due to controllable mechanical properties and tailorable biodegradation. Here, silica/chitosan hybrid scaffolds were fabricated through the sol-gel method with a freeze drying process. 3-Glycidoxypropyl trimethoxysilane (GPTMS) and tetraethylorthosilicate (TEOS) were used as the covalent inorganic/organic coupling agent and the separate inorganic source, respectively. Hybrid scaffolds with various inorganic/organic weight ratios (I/Os) and molar ratios of chitosan and GPTMS (GCs) were examined and compared in this study. FTIR showed that higher GPTMS content resulted in the increased covalent cross-linking of the chitosan and the silica network in hybrids. Compression testing indicated that increasing the GPTMS content greatly improved the compressive strength of scaffold. LIVE/DEAD assay showed that enhanced cytocompatibility was obtained as the silica content increased. Therefore, the results confirmed that the two parameters I/O and GC can largely influence the scaffold performance, which can be used to tailor the hybrid properties.

DNA methylation has emerged as a crucial regulator of chondrocyte dedifferentiation, which severely compromises the outcome of autologous chondrocyte implantation (ACI) treatment for cartilage defects. However, the full-scale DNA methylation profiling in chondrocyte dedifferentiation remains to be determined. Here, we performed a genome-wide DNA methylation profiling of dedifferentiated chondrocytes in monolayer culture and chondrocytes treated with DNA methylation inhibitor 5-azacytidine (5-AzaC). This research revealed that the general methylation level of CpG was increased while the COL-1A1 promoter methylation level was decreased during the chondrocyte dedifferentiation. 5-AzaC could reduce general methylation levels and reverse the chondrocyte dedifferentiation. Surprisingly, the DNA methylation level of COL-1A1 promoter was increased after 5-AzaC treatment. The COL-1A1 expression level was increased while that of SOX-9 was decreased during the chondrocyte dedifferentiation. 5-AzaC treatment up-regulated the SOX-9 expression while down-regulated the COL-1A1 promoter activity and gene expression. Taken together, these results suggested that differential regulation of the DNA methylation level of cartilage-specific genes might contribute to the chondrocyte dedifferentiation. Thus, the epigenetic manipulation of these genes could be a potential strategy to counteract the chondrocyte dedifferentiation accompanying in vitro propagation. J. Cell. Physiol. 232: 1708-1716, 2017. © 2016 Wiley Periodicals, Inc.

Developed in recent years, low-temperature deposition manufacturing (LDM) represents one of the most promising rapid prototyping technologies. It is not only based on rapid deposition manufacturing process but also combined with phase separation process. Besides the controlled macropore size, tissue-engineered scaffold fabricated by LDM has inter-connected micropores in the deposited lines. More importantly, it is a green manufacturing process that involves non-heating liquefying of materials. It has been employed to fabricate tissue-engineered scaffolds for bone, cartilage, blood vessel and nerve tissue regenerations. It is a promising technology in the fabrication of tissue-engineered scaffold similar to ideal scaffold and the design of complex organs. In the current paper, this novel LDM technology is introduced, and its control parameters, biomedical applications and challenges are included and discussed as well.

The transforming growth factor β1 (TGFβ1) plays an important role in cartilage development. However, whether TGFβ1 stimulates chondrocyte proliferation and cartilage regeneration in osteoarthritis (OA) remains elusive, especially in the context of different treatment and tissue environments. In the present study, we investigated the role of TGFβ1 in human chondrocyte culture in vitro, focusing on the morphological change of chondrocytes and the expression of angiogenic factors upon TGFβ1 stimulation. We found increased expression of biomarkers indicating chondrocyte hypertrophy and the chondrocytes aggregated to form networks when they were treated with TGFβ1. DNA microarray analysis revealed significantly increased expression of genes related to blood vessel formation in TGFβ1 treatment group compared to control group. Matrigel assay further demonstrated that chondrocytes had the potential to form network-like structure. These results suggested that TGFβ1 induces the hypertrophic change of chondrocytes culture in vitro and induce expression of angiogenic biomarkers. Therefore, application of TGFβ1 for chondrocyte culture in practice should be considered prudentially and targeting TGFβ1 or relevant receptors to block the signaling pathway might be a strategy to prevent or alleviate progression of osteoarthritis.

BACKGROUND:By mixing technology, various materials are mixed and complemented each other to enhance heat sensitivity, mechanical properties, viscoelasticity of the materials and improve their biocompatibility, biodegradability and other biomedical properties.OBJECTIVE:To prepare polyvinyl alcohol (PVA)/chitosan (CS) porous hydrogel and to observe its effects on the repair of articular cartilage defects.METHODS: PVA, CS and polysorbate-80 were used as raw materials to prepare the hydrogels at the PVA/CS ratio of 5:5, 6:4, 7:3, 8:2 and 9:1 by freezing-thawing cycle and emulsification-frostice phase separation, and the physical and chemical properties, mechanical properties, and biocompatibility of the hydrogels were detected to find out the best PVA/CS ratio. Eighteen New Zealand rabbits were enroled to make models of bilateral articular cartilage defects, and then randomized into three groups: blank control group; control group; experimental group. The prepared hydrogel was compounded with rabbit bone marrow mesenchymal stem cells (BMSCs) and then implanted into the rabbit articular cartilage defect in the experimental group, the PVA/CS hydrogel was implanted in the control group, and nothing was implanted in the blank control group. After 12 weeks, the animals were killed and the repairing effect was observed by gross observation and histological examination.RESULTS AND CONCLUSION:The PVA/CS porous composite hydrogel was successfully prepared, and PVA:CS=6:4 was the best in the presence of good mechanical properties, stable physical and chemical properties, and water content≥ 90%. In additional, scanning electron microscopy showed a porous network structure, with the porosity≥ 90%. The results of cell counting kit-8 assay and the results of cell death and survival showed that the hydrogel was non-cytotoxic and beneficial to the cell proliferation. In the blank control group, knee articular cartilage defects were not repaired within 12 weeks after surgery, showing significant granulation tissue filling. In the control group, knee articular cartilage defects were full of cartilage-like tissues with no smooth surface, but there were a great amount of chondrocyte-like cells. In the experimental group, knee articular cartilage defects were well repaired and full of a great amount of chondrocytes with the smooth surface. To conclude, the PVA/CS porous composite hydrogel could repair articular cartilage defects as an ideal tissue-engineered cartilage material.

BACKGROUND:Estrogen deficiency is closely related to the development of menopausal arthritis. Estrogen replacement therapy (ERT) shows a protective effect against the osteoarthritis. However, the underlying mechanism of this protective effect is unknown. This study aimed to determine the role of miR-140 in the estrogen-dependent regulation of MMP-13 in human chondrocytes.METHODS:Primary human articular chondrocytes were obtained from female OA patients undergoing knee replacement surgery. Normal articular chondrocytes were isolated from the knee joints of female donors after trauma and treated with interleukin-1 beta (IL-1β). Gene expression levels of miR-140, MMP-13, and ADAMTS-5 were detected by quantitative real-time PCR (qRT-PCR). miR-140 levels were upregulated or downregulated by transfecting cells with a miRNA mimic and inhibitor, respectively, prior to treatment with IL-1β. MMP-13 expression was then evaluated by Western blotting and immunofluorescence. Luciferase reporter assays were performed to verify the interaction between miR-140 and ER.RESULTS:17-β-estradiol (E2) suppressed MMP-13 expression in human articular chondrocytes. miR-140 expression was upregulated after estrogen treatment. Knockdown of miR-140 expression abolished the inhibitory effect of estrogen on MMP-13. In addition, the estrogen/ER/miR-140 pathway showed an inhibitory effect on IL-1β-induced cartilage matrix degradation.CONCLUSIONS:This study suggests that estrogen acts via ER and miR-140 to inhibit the catabolic activity of proteases within the chondrocyte extracellular matrix. These findings provide new insight into the mechanism of menopausal arthritis and indicate that the ER/miR-140 signaling pathway may be a potential target for therapeutic interventions for menopausal arthritis.

Purpose: This study was conducted to investigate the feasibility and clinical potential of using the 3D printing technology (3DPT) versus typical strategy (thin-layer CT scan) for the treatment of complicated proximal humeral fractures (PHFs) in old people.Methods: Sixty-six old patients age ranging from 61 to 76 years with persistent complicated PHFs were randomly assigned to two groups as per the controlled randomization table (34 cases in the test group and 32 cases in the control group). In the test group, 3DPT was applied to build the 3D facture model of a patient, according to the data acquired from the thin-layer CT scan and subsequently processed with Mimics software. This helped to confirm the diagnosis, design the individual operation plan, simulate the surgical procedures and perform the surgery as plan. In the control group, only thin-layer CT scan was applied for the design of the operation plan prior to the surgery. Here, parameters including surgery duration, blood loss volume during surgery, the number of fluoroscopy, time to union were statistically analyzed for two groups after the operation. The screw lengths designed before the surgery and measured during the surgery were compared.Results: The 3D PHF model generated using 3DPT was able to provide the visual display and omnidirectional observation of the direction and severity of the fracture dislocation, which facilitated preoperative diagnosis, operation planning and design, data measurement, preselection of internal fixator and surgical outcome simulation. According to the follow-up ranging from 12 similar to 28 months for the 66 patients, the results showed no significant difference in time to union between the two groups (P>0.05). Apart from that, less surgery duration, less blood loss during surgery, less number of fluoroscopy can be observed compared with the control group (P<0.05).Conclusions: In this study, 3DPT showed great clinical feasibility of the treatment of complicated PHFs. The 3D-print PHF model had the ability to clearly display the fracture and thus was useful to determine the fracture classification and the magnitude of fracture injury. It benefited surgeons to gain a better understanding of complicated PHFs, design a most suitable operation plan prior to surgery and facilitate the doctor-patient communication. This therefore enabled the reduction of intraoperative injury and the optimization of surgical outcomes. (C) 2016 Elsevier Masson SAS. All rights reserved.

This study aims to develop an effective method to control motile microorganisms and enable their manipulation as functional 'live micro/nano robots'. A novel strategy based on Fe3 O4 nanoparticle-doped alginate hydrogel is developed to fashion an artificial extracellular matrix (ECM) for microbial cells (e.g., Saccharomyces cerevisiae and Flavobacterium heparinum). During this strategy, a single layer of alginate hydrogel is coated around the microbial cells doped with Fe3 O4 nanoparticles to form the alg-mag-cells. Transmission electron microscopy shows that Fe3 O4 nanoparticles are uniformly distributed in the hydrogel shell. Together with maintaining the cell activity and metabolism, the hydrogel coated microbial cells demonstrate high magnetic responsiveness in an external magnetic field and are able to form micro-scaled patterns using the magnetic template designed in this study. This strategy provides a building block to fabricate advanced biological models, medical therapeutic products, and non-medical biological systems using different microorganisms.

This study was conducted to investigate the feasibility and clinical potential of using the 3D printing technology (3DPT) versus typical strategy (thin-layer CT scan) for the treatment of complicated proximal humeral fractures (PHFs) in old people. Sixty-six old patients age ranging from 61 to 76 years with persistent complicated PHFs were randomly assigned to two groups as per the controlled randomization table (34 cases in the test group and 32 cases in the control group). In the test group 3DPT was applied to build the 3D facture model of a patient, according to the data acquired from the thin-layer CT scan and subsequently processed with Mimics software. This helped to confirm the diagnosis, design the individual operation plan, simulate the surgical procedures and perform the surgery as plan. In the control group, only thin-layer CT scan was applied for the design of the operation plan prior to the surgery. Here parameters including surgery duration, blood loss volume during surgery, the number of fluoroscopy, time to union were statistically analyzed for two groups after the operation. The screw lengths designed before the surgery and measured during the surgery were compared. The 3D PHF model generated using 3DPT was able to provide the visual display and omni-directional observation of the direction and severity of the fracture dislocation, which facilitated preoperative diagnosis, operation planning and design, data measurement, pre-selection of internal fixator and surgical outcome simulation. According to the follow-up ranging from 12∼28 months for the 66 patients, the results showed no significant difference in time to union between the two groups (P > 0.05). Apart from that, less surgery duration, less blood loss during surgery, less number of fluoroscopy can be observed compared with the control group (P < 0.05). In this study 3DPT showed great clinical feasibility of the treatment of complicated PHFs. The 3D-print PHF model had the ability to clearly display the fracture and thus was useful to determine the fracture classification and the magnitude of fracture injury. It benefited surgeons to gain better understanding of complicated PHFs, design a most suitable operation plan prior to surgery and facilitate the doctor–patient communication. This therefore enabled the reduction of intraoperative injury and the optimization of surgical outcomes.

Inorganic/organic sol-gel hybrids have nanoscale co-networks of organic and inorganic components that give them the unique potential of tailored mechanical properties and controlled biodegradation in tissue engineering applications. Here, silica/chitosan hybrid scaffolds with oriented structures were fabricated through the sol-gel method with a unidirectional freeze casting process. 3-Glycidoxypropyl trimethoxysilane (GPTMS) was used to obtain covalent inorganic/organic coupling. Process variables were investigated such as cooling rate, GPTMS and inorganic content, which can be used to tailor the mechanical properties and hybrid chemical coupling. Structural characterization and dissolution tests confirmed the covalent cross-linking of the chitosan and the silica network in hybrids. The scaffolds had a directional lamellar structure along the freezing direction and a cellular morphology perpendicular to the freezing direction. Compression testing showed that the scaffolds with 60 wt% organic were flexible and elastomeric perpendicular to the freezing direction whilst behaving in an elastic-brittle fashion parallel to the freezing direction. The compressive strengths are about one order of magnitude higher in the latter direction reaching values of the order of 160 kPa. This behaviour provides potential for clinicians to be able to squeeze the materials to fit tissue defect sites while providing some mechanical support from the other direction.

Current materials used for bone regeneration are usually bioactive ceramics or glasses. Although they bond to bone, they are brittle. There is a need for new materials that can combine bioactivity with toughness and controlled biodegradation. Sol-gel hybrids have the potential to do this through their nanoscale interpenetrating networks (IPN) of inorganic and organic components. Poly(γ-glutamic acid) (γ-PGA) was introduced into the sol-gel process to produce a hybrid of γ-PGA and bioactive silica. Calcium is an important element for bone regeneration but calcium sources that are used traditionally in the sol-gel process, such as Ca salts, do not allow Ca incorporation into the silicate network during low-temperature processing. The hypothesis for this study was that using calcium methoxyethoxide (CME) as the Ca source would allow Ca incorporation into the silicate component of the hybrid at room temperature. The produced hybrids would have improved mechanical properties and controlled degradation compared with hybrids of calcium chloride (CaCl2), in which the Ca is not incorporated into the silicate network. Class II hybrids, with covalent bonds between the inorganic and organic species, were synthesised by using organosilane. Calcium incorporation in both the organic and inorganic IPNs of the hybrid was improved when CME was used. This was clearly observed by using FTIR and solid-state NMR spectroscopy, which showed ionic cross-linking of γ-PGA by Ca and a lower degree of condensation of the Si species compared with the hybrids made with CaCl2 as the Ca source. The ionic cross-linking of γ-PGA by Ca resulted in excellent compressive strength and reduced elastic modulus as measured by compressive testing and nanoindentation, respectively. All hybrids showed bioactivity as hydroxyapatite (HA) was formed after immersion in simulated body fluid (SBF).

Understanding the distribution of critical elements (e.g. silicon and calcium) within silica-based bone scaffolds synthesized by different methods is central to the optimization of these materials. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been used to determine this information due to its very high surface sensitivity and its ability to map all the elements and compounds in the periodic table with high spatial resolution. The SIMS image data can also be combined with depth profiles to construct three-dimensional chemical maps. However, the scaffolds have interconnected pore networks, which are very challenging structures for the SIMS technique. To overcome this problem two experimental methodologies have been developed. The first method involved the use of the focused ion beam technique to obtain clear images of the regions of interest and subsequently mark them by introducing fiducial marks; the samples were then analysed using the ToF-SIMS technique to yield the chemical analyses of the regions of interest. The second method involved impregnating the pores using a suitable reagent so that a flat surface could be achieved, and this was followed by secondary ion mapping and 3D chemical imaging with ToF-SIMS. The samples used in this work were sol-gel 70S30C foam and electrospun fibres and calcium-containing silica/gelatin hybrid scaffolds. The results demonstrate the feasibility of both these experimental methodologies and indicate that these methods can provide an opportunity to compare various artificial bone scaffolds, which will be of help in improving scaffold synthesis and processing routes. The techniques are also transferable to many other types of porous material.

Inorganic sol-gel solutions were electrospun to produce the first bioactive three-dimensional (3-D) scaffolds for bone tissue regeneration with a structure like cotton-wool (or cotton candy). This flexible 3-D fibrous structure is ideal for packing into complex defects. It also has large inter-fiber spaces to promote vascularization, penetration of cells and transport of nutrients throughout the scaffold. The 3-D fibrous structure was obtained by electrospinning, where the applied electric field and the instabilities exert tremendous force on the spinning jet, which is required to be viscoelastic to prevent jet break up. Previously, polymer binding agents were used with inorganic solutions to produce electrospun composite two-dimensional flbermats, requiring calcination to remove the polymer. This study presents novel reaction and processing conditions for producing a viscoelastic inorganic sol-gel solution that results in fibers by the entanglement of the intermolecularly overlapped nanosilica species in the solution, eliminating the need for a binder. Three-dimensional cotton-wool-like structures were only produced when solutions containing calcium nitrate were used, suggesting that the charge of the Ca2+ ions had a significant effect. The resulting bioactive silica fibers had a narrow diameter range of 0.5-2 mu m and were nanoporous. A hydroxycarbonate apatite layer was formed on the fibers within the first 12 h of soaking in simulated body fluid. MC3T3-E1 preosteoblast cells cultured on the fibers showed no adverse cytotoxic effect and they were observed to attach to and spread in the material. Crown Copyright (C) 2014 Published by Elsevier Ltd. on behalf of Acta Materialia Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).