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With a long history and rich cultural connotation, Luoshan Shadow puppet has become an intangible cultural heritage of China. However, in the midst of modernisation, there are some problems with the design of Luoshan shadow creative products. In this paper, we examined an overview of research into the Luoshan shadow creative products. These products have been criticised for their lack of creativity in all design activities, which has hindered their development. In order to obtain more reliable results, we chose to conduct an extensive questionnaire survey in China. A descriptive analysis of all user data from these questionnaires was carried out. 379 participants' data showed the theme of this study; i. problems of stylistic design; ii. problems of lack of distinctiveness; and iii. problems of overpricing. These findings are beneficial to further research on the innovative design of Luoshan shadow creative products.

The correlation between dietary inflammation index (DII) and rheumatoid arthritis (RA) has been found, but the effect of confounding factors is not considered. This study aims to further explore the association between DII and RA risk by taking the Americans as the research object.The data from the 2005-2018 National Health and Nutrition Examination Survey (NHANES) database included 1819 self-reported RA individuals and 8602 non-RA individuals. The analytical methods include logistic regression, additive model, smooth curve fitting, and the recursive algorithm.There was a positive correlation between DII and RA in Americans (β = 1.068, 95% CI = 1.026 to 1.111, P = 0.001). This result was still presented in the subgroup analysis, including age less than 50 years, female, other Hispanics, BMI ≥ 25, and federal poverty rate 185%, and it was more pronounced in smokers. The results show that the superposition of DII and other risk factors would increase the risk of RA (β 1.068). In addition, individuals with RA are inadequate in intake of anti-inflammatory foods, in line with the Mediterranean diet.The inflammatory potential of the diet is positively correlated with the risk of RA, and has a superimposed effect with other risk factors, increasing the probability of the risk of disease. These results emphasize that reducing the intake of pro-inflammatory foods may be an effective measure to prevent the onset of rheumatoid arthritis. However, eating anti-inflammatory foods exclusively is not the best option. Intaking some pro-inflammatory foods like protein, energy, and total saturated acids may be necessary to maintain the physiological function of the human body. Key Points • Dietary inflammation index (DII) is positively correlated with RA risk. • When DII and other risk factors appear at the same time, the effects of the two will be superimposed on each other, increasing the risk of RA. • When the DII is the same, Hispanic has a higher incidence of RA. • Among the pro-inflammatory foods, the intake of protein, energy, and saturated fatty acids is still required by RA patients.

To solve the problems of the low target-positioning accuracy and weak algorithm robustness of target-dosing robots in greenhouse environments, an image segmentation method for cucumber seedlings based on a genetic algorithm was proposed. Firstly, images of cucumber seedlings in the greenhouse were collected under different light conditions, and grayscale histograms were used to evaluate the quality of target and background sample images. Secondly, the genetic algorithm was used to determine the optimal coefficient of the graying operator to further expand the difference between the grayscale of the target and background in the grayscale images. Then, the Otsu algorithm was used to perform the fast threshold segmentation of grayscale images to obtain a binary image after coarse segmentation. Finally, morphological processing and noise reduction methods based on area threshold were used to remove the holes and noise from the image, and a binary image with good segmentation was obtained. The proposed method was used to segment 60 sample images, and the experimental results show that under different lighting conditions, the average F1 score of the obtained binary images was over 94.4%, while the average false positive rate remained at about 1.1%, and the image segmentation showed strong robustness. This method can provide new approaches for the accurate identification and positioning of targets as performed by target-dosing robots in a greenhouse environment.

This letter presents a voltage-controlled oscillator (VCO) with an extra cross-coupling path realized by a novel transformer-based resonator. The extra cross-coupling can enhance the negative $G_{m}$ and loop gain of the cross-coupled pair. The triode time of the active device during the oscillation will be reduced, resulting in better phase noise (PN) and shorter startup time than the conventional $LC$ -VCO. Fabricated in a CMOS 65-nm process, the proposed VCO consumes 0.9 mW average dc power under a voltage supply of 0.38 V. The 1-MHz offset PN can reach −107.15 dBc/Hz at 8.38 GHz, and the corresponding figure of merit (FoM) is −186.75 dBc/Hz. The VCO’s bandwidth is 34.1% ranging from 6.26 to 8.84 GHz, which corresponds an FoMT of −197.41 dBc/Hz. Thanks to the compact layout of the transformer, the VCO occupies only 0.055 mm2 core area, resulting in a −199.35 dBc/Hz FoMA.

With the rapid increase in the penetration of wind power in recent years, wind power ramp events (WPREs) have become the main factors affecting the safety and stability of electric power systems. Accurate detection of ramp events could help power systems better manage the extreme events and reduce economic losses. The previous ramp detection methods are either too complex to implement that influence the computing efficiency, or based on the value of points which cannot completely reflect the trend of data segments and lead to a decrease of accuracy. Based on the above problems and the on-site requirements, this paper proposes a novel improved dynamic swinging door algorithm (ImDSDA) to optimise the state-of-the-art in WPREs detection. Firstly, the swinging door algorithm (SDA) is used to extract ramp segments. Secondly, the dynamic programming method is used for ramp trend identification and segment combination. Finally, raw data obtained from three real-world wind farms in Hubei, China were applied to validate the performance of the proposed ImDSDA. The detection results show that the ImDSDA is more accurate and efficient than the traditional detection methods and could be a feasible option for WPRE detection in power systems.

Summary Purpose The purpose of our study was to introduce and validate a metal-free, reproducible and reliable mouse model of anterior cruciate ligament (ACL) reconstruction (ACLR) surgery as an effective tool for a better understanding of molecular mechanisms of graft-tunnel healing after ACLR. Methods A total of 150 C57BL/6 mice were randomly allocated into five Groups: Group 1 (mice with intact ACL), Group 2–4 (mice underwent modified ACLR surgery and sacrificed 1-, 2-, and 4-weeks after surgery), and Group 5 (mice underwent unmodified ACLR surgery and sacrificed 4 weeks after surgery). Micro-computed tomography (CT), biomechanical histological as well as immunohistochemical (IHC) analyses were performed to characterize the modified ACLR. Results Micro-CT analysis demonstrated there is a non-significant increase in BV/TV and BMD of the bone tunnel during the tendon-to-bone healing following ACLR. Biomechanical tests showed that the mean load-to-failure forces of Group 3 and 4 are equal to 31.7% and 46.0% of that in Group 1, while the stiffness was 33.1% and 57.2% of that of Group 1, respectively. And no obvious difference in biomechanical parameters was found between Group 4 and 5. Histological analysis demonstrated that formation of fibrovascular tissue in the tibial tunnel and aperture in Groups 4 and 5 and direct junction appeared between tendon graft and tunnel both in Groups 4 and 5. IHC results showed that there are gradually enhanced expression of Patched1, Smoothened and Gli2 concomitant with decreased Gli3 protein in the tendon-bone interface during the tendon-bone healing process. Conclusion We introduced a metal-free, reproducible and reliable mouse model of ACLR compared to the unmodified ACLR procedure, and characterized the expression pattern of key molecules in Ihh signaling during the graft healing process. The translational potential of this article In the present study we introduced and validated, for the first time, a metal-free, reproducible and reliable ACLR mouse model, which could be used to investigate the detailed molecular mechanisms of graft-tunnel healing after ACLR. We also explored new strategies to promote the healing of tendon-to-bone integration.

Background/objective. LIM and LIM zinc finger domain containing 2 (LIMS2) is one of the two members of LIMS family, which plays crucial roles in regulating cell-extracellular matrix adhesion and cell motility. Here, we explored the expression and methylation levels of LIMS2 in osteosarcoma (OS) and the role of LIMS2 in OS progression. Methods. GEO, GEPIA, and UALCAN databases were used to assess LIMS2 expression in OS. UALCAN and CCLE databases were applied to assess the methylation levels of LIMS2 in OS tissues and cells, which was verified in OS cells using the methylation specific PCR. The effects of LIMS2 on regulating OS cell growth, migration and invasion were determined by CCK-8, Edu staining, and transwell chambers, respectively. The role of LIMS2 in the activation of MAPK signaling was assessed using western blotting assay in OS cells. Results. LIMS2 expression was declined in OS tissues and cells, while its methylation level was increased. The low expression of LIMS2 was associated with shorter overall survival and disease-free survival. Overexpression of LIMS2 inhibited cell growth, migration, and invasion and decreased the levels of p-ERK/ERK, p-P38/P38, and p-JNK/JNK. Conclusion. LIMS2 expression was decreased in OS, which was associated with hypermethylation level and poor prognosis. LIMS2 overexpression inhibited OS cell growth and migration, which may be caused by the suppression of MAPK signaling.

Thermal energy storage with phase change materials (PCM) is of great significance for increasing energy efficiency and energy saving. In this paper, a novel capric acid-myristyl alcohol/expanded perlite composite PCM (CPCM) with two phase change peaks were prepared. The capric acid-myristyl alcohol (CM) were synthesized by capric acid (CA) and myristyl alcohol (MA) (WCA and WMA = 9:1). Three kinds of expanded perlites (EP) were employed as support materials in the prepared CPCM. The chemical structure, morphology, thermal properties, leakage and thermal energy storage performance of the CM/EPPCM were investigated. The results indicate that the CM/EPPCM has a good thermal stability and leakage-proof performance. In addition, the thermal characteristics of the CM/EPPCM were investigated using a building model. The experimental results shows that the application of the CM/EPPCM for building envelope can effectively reduce temperature fluctuation and improve the thermal comfort of the building.

The contamination of pesticide residues in Oolong tea has raised much concern in recent years. The objective of this study was to synthesize gold nanoparticles (AuNPs) and develop surface-enhanced Raman spectroscopy (SERS) methods for detection and quantification of pesticides in Oolong tea. Facile synthesis of spherical and monodispersed AuNPs with an average diameter of 15 nm was achieved, which induced strong electromagnetic fields in SERS analysis. AuNP substrates were employed for rapid detection and quantification of carbendazim in Tieguanyin Oolong tea. Partial least squares (PLS) analysis and leave-one-out cross validation were utilized in spectral data analysis. The PLS results for Oolong tea samples were obtained: R value = 0.964; the detection limit = 100 μg/kg. These results demonstrate that SERS coupled with gold nanoparticle substrate is a simple, rapid, and sensitive analytical tool for measurement and quantification of carbendazim residues in Oolong tea.

Selenium-substituted HA (Se/HA) particles with special micro/nanohybrid morphologies were prepared hydrothermally. With increasing Se substitution, Se/HA transitioned from a ribbon-like morphology, to a radical form, and further to microspheres (diameters: ∼10 μm) assembled from nanosheets (thickness: 2–5 μm; length and width: ∼100 nm). Chemical analyses confirmed increasing incorporation of Se in Se/HA particles, with increasing Se/(Se + P) molar ratio of the raw materials for their synthesis. Rat calvarial osteoblasts (RCOBs) adhered and spread on the Se/HA particles with Se/(Se + P) ratios ranging 0–0.05. Particles synthesized at Se/(Se + P) = 0.05 showed increased alkaline phosphatase activity of bone marrow mesenchymal stem cells, likely due to their micro/nano-sized surface morphology. Se released from particles effectively inhibited human osteosarcoma cell growth by inducing apoptosis. Therefore, Se/HA prepared at Se/(Se + P) = 0.05 presented a potential material for bone defect repair after surgical tumor removal, offering safety, increased osteogenic differentiation of stem cells, and inhibition of tumor cell growth. These results also suggest that trace element substitution is an effective approach to simultaneously imparting desirable microstructures and biological functions to bio-ceramics.

Researches have revealed the vital roles of the generated immune environment via the response of immune cells growing on biomaterial surfaces in the bone healing process. HAS and novel constructed microgrooved patterns of HAS (HAS-G) are widely used as biocompatible ceramic, especially as a mimic of the natural bone matrix. However, it is unclear whether osteoimmune response induced by HAS and HAS-G affects the osteogenic differentiation of bone marrow stromal cells (BMSCs). RAW264.7 cells were seeded on different surface of materials and cytokines released by macrophages were detected by enzyme-linked immunosorbent assay. The cell viability and mitochondrial function of macrophages seeded on different surface of materials were detected. Then, the effects of modified inflammatory microenvironment by macrophages on osteogenesis of BMSCs were measured by performing ALP staining, Alizarin Red S staining, and western blot. We confirmed that HAS-G is more favorable for RAW cell attaching and subsequently regulated the expression and release of cytokines/chemokines. Decrease in interleukin-6 (IL-6) release was further confirmed for contributing significantly to improve mitochondrial function in RAW cells. HAS-G-conditioned medium promoted osteogenic differentiation in BMSCs and was reversed by IL-6 addition. Decrease in IL-6 contributes to downregulation of miR-214 and subsequently upregulated p38/JNK pathway, which is potentially contributes to osteogenic promotion by HAS-G. This study is the first report to reveal the effects of HAS-G on osteogenesis via immune response, which could lead to a new insight into novel material for the advantage of biomaterials for tissue engineering applications.

Pullulanase is a starch-debranching enzyme that is generally employed to efficiently break down starch for the production of high-glucose syrup. Acidic adaptation of pullulanases is of special interest. In this study, we conducted protein engineering to improve the acidic adaptation of Bacillus acidopullulyticus pullulanase (BaPul) and used a hydrogen-bond-based approach to identify promising residues that may change the deprotonation constants (pKa) of the catalytic residues. A total of 19 amino acids were selected for mutation according to the crystal structure of BaPul. The pH optimum of the L627R mutant shifted from 5.0 to 4.0, and its relative activity at pH 4.0 was 117% that of the wide-type enzyme. The improved efficacy of the L627R mutant at pH 4.0 was confirmed by kinetic parameters and pKa prediction. Moreover, the L627R mutant exhibited increased tolerance against acid-mediated denaturation, and its maximum d -glucose content (97.4%) was obtained after 40 h incubation, which is shorter by 10 h compared with the time required by the wide-type enzyme to produce a comparable amount of the monosaccharide. The L627R mutant may be suitable for industrial application because its shortened reaction time translates to reduced energy consumption.

Obesity is closely associated with low-bone-mass disorder. Discoidin domain receptor 2 (DDR2) plays essential roles in skeletal metabolism, and is probably involved in fat metabolism. To test the potential role of DDR2 in fat and fat-bone crosstalk, Ddr2 conditional knockout mice (Ddr2Adipo) were generated in which Ddr2 gene is exclusively deleted in adipocytes by Adipoq Cre. We found that Ddr2Adipo mice are protected from fat gain on high-fat diet, with significantly decreased adipocyte size. Ddr2Adipo mice exhibit significantly increased bone mass and mechanical properties, with enhanced osteoblastogenesis and osteoclastogenesis. Marrow adipocyte is diminished in the bone marrow of Ddr2Adipo mice, due to activation of lipolysis. Fatty acid in the bone marrow was reduced in Ddr2Adipo mice. RNA-Seq analysis identified adenylate cyclase 5 (Adcy5) as downstream molecule of Ddr2. Mechanically, adipocytic Ddr2 modulates Adcy5-cAMP-PKA signaling, and Ddr2 deficiency stimulates lipolysis and supplies fatty acid for oxidation in osteoblasts, leading to the enhanced osteoblast differentiation and bone mass. Treatment of Adcy5 specific inhibitor abolishes the increased bone mass gain in Ddr2Adipo mice. These observations establish, for the first time, that Ddr2 plays an essential role in the crosstalk between fat and bone. Targeting adipocytic Ddr2 may be a potential strategy for treating obesity and pathological bone loss simultaneously.

This paper presents a wideband low-noise-amplifier using current-reused technique with transformer feedback for wideband input/output matching; simultaneously covering two major 5G millimeter frequency bands (28-GHz, 37-GHz). The LNA is based on a 2-stage current-reused common source and common source (CS-CS), which integrated gate-drain transformer feedback for achieving wideband input/output matching. The LNA, which operates from 17 GHz up to 48 GHz with 95% 3-dB bandwidth (17-48 dB) and 74.6% 1-dB bandwidth (21-46 GHz), exhibits maximum gain of 18.7 dB and minimum noise Figure of 4.3 dB. The LNA is designed in a 65-nm CMOS technology of TSMC and only dissipates 16 mW from 1.2-V supply voltage.

A 28-GHz CMOS image-reject mixer (IRM) for 5G applications with an intermediate frequency (IF) of 3.6 GHz is presented. The IRM employs a folded down-conversion mixer for low voltage operation. PMOS as switch stage in IRM requires a lower LO power than NMOS. An active polyphase filter (APPF) with better phase balance than passive polyphase filter (PPPF) is applied to realize improved image rejection at IF. A two-stage PPPF is a better choice to convert the differential LO signals to differential and quadrature signals than APPF. The IRM exhibits 12.5 ± 1.5 dB conversion gain (CG) and the noise figure (NF) is 12.7 dB at IF 3.6 GHz. The maximum image-reject ratio (IRR) at fixed LO 23 GHz is 57 dB. The IRR of better than 40 dB is achieved for the IF from 2 to 4 GHz. The IRM is designed in a 65-nm CMOS technology and only dissipates 15 mW from a 1-V supply voltage.

Chondrogenesis and subsequent osteogenesis of mesenchymal stem cells (MSCs) and angiogenesis at injured sites are crucial for bone fracture healing. Amygdalin, a cyanogenic glycoside compound derived from bitter apricot kernel, has been reported to inhibit IL-1β-induced chondrocyte degeneration and to stimulate blood circulation, suggesting a promising role of amygdalin in fracture healing. In this study, tibial fractures in C57BL/6 mice were treated with amygdalin. Fracture calluses were then harvested and subjected to radiographic, histological, and biomechanical testing, as well as angiography and gene expression analyses to evaluate fracture healing. The results showed that amygdalin treatment promoted bone fracture healing. Further experiments using MSC-specific transforming growth factor- (TGF-) β receptor 2 conditional knockout (KO) mice (Tgfbr2Gli1-Cre) and C3H10 T1/2 murine mesenchymal progenitor cells showed that this effect was mediated through TGF-β/Smad signaling. We conclude that amygdalin could be used as an alternative treatment for bone fractures.

Magnesium (Mg) is an emerging degradable metal for orthopaedics and its degradation product, Mg2+, has been reported to positively affect osteogenesis. Porous hydroxyapatite (HA) has been extensively studied for bone regeneration, but its slow degradation is an important factor limiting its uses. Therefore, we studied the combination of Mg and HA to integrate the advantages of the two materials while circumventing their disadvantages, with the aim of determining the optimum Mg/HA ratio. Surface-treated Mg particles (Φ ∼ 1.3 mm) and HA spheres (Φ ∼ 1.4 mm) were mixed at 0, 10, 30, and 50% (v/v). The mixtures were filled in titanium cages to form complexes. In vitro biocompatibility was studied by culturing MC3T3-E1 osteoblasts in extracts prepared from the complexes. In vivo biocompatibility and osteoinduction were evaluated by implantation in canine dorsal muscles. All complexes were not cytotoxic to the osteoblasts, regardless of the Mg proportion. Eight weeks after implantation in muscles, complexes containing 50% Mg showed inflammatory cell infiltration, whereas all other groups showed no evident inflammatory response. Additionally, complexes containing 30% Mg had significantly higher new bone area percentage (BV/TV), trabecular number (Tb.N), and bone growth rate than those containing 0, 10%, or 50% Mg. In comparison, complexes containing 50% Mg had significantly lower BV/TV and Tb.N than all other groups, and also had a significantly lower bone growth rate than those containing 10% or 30% Mg. Sixteen weeks after implantation, the trend in bone formation was similar to that observed at week 8. This shows that metallic Mg affected the osteoinductivity of HA-Mg complexes in a dose-dependent manner. In the present model, 30% Mg induced optimum osteoinduction. More studies are needed to explore the optimum Mg fractions for other implantation sites or forms of use.

The repair of large maxillofacial bony defects using regular scaffolds is restricted by the osteogenic effect. It was postulated that a novel porous hydroxyapatite (HA) scaffolds with a 25–30 µm groove structure (HAG) may counter this limitation. In this study we evaluated the biocompatibility of HAG scaffolds both in vitro and in vivo in beagle dogs by investigating the enhancement of scaffolds bioactivity and osteogenesis. Compared with a regular HA scaffolds, the HAG scaffolds significantly promoted human placenta-derived mesenchymal stem cell (hPMSC) osteogenic differentiation and the maturation of osteoblasts. This is achieved by increasing protein adsorption, as well as promoting directed growth and expression of osteogenic genes in vitro. The compressive strength of HAG scaffolds was significantly greater than HA in both dorsal muscle and the mandibular distraction area after in vivo implantation, with hematoxylin and eosin staining demonstrating new bone formation and vasculogenesis. Immunochemical staining and micro-CT scanning demonstrated increased expression of osteogenic factors (BMP2, OCN and COL-1) and bone density in the HAG scaffolds compared with HA. Based on the above results, we conclude that HAG scaffolds that have a groove structure induce greater osteogenesis and possess improved ostoegenesis which could be utilized in the clinical treatment.

A novel composite phase change material (PCM) used as exterior and interior envelop materials for passive solar energy utilization was synthesized and investigated in this paper. Eutectic hydrated salt (EHS) consisted sodium carbonate decahydrate (SCD) and disodium hydrogen phosphate dodecahydrate (DHPD) with a mass ratio of 5:5 was prepared as the PCM with its phase change temperature of 26.94 °C and phase change enthalpy of 198.44 J/g while three different kinds of expanded perlites (EPs) were pretreated as supporting materials. According to the results of leakage tests and volume enthalpy, 70%EHS/EP2 was chosen as the form-stable phase change materials (FSPCM) to fabricate phase change insulation panels. The thermal conductivity with different temperature of 70%EHS/EP2 was measured and the maximum was 0.44 W/(m K) when the testing temperature was 25 °C. In addition, the heat storage and release results of 70%EHS/EP2 indicated that there was no supercooling. The thermogravimetric analysis and thermal cycling tests were performed and the results indicated that 70%EHS/EP2 had a good thermal stability and reliability. The heat transfer properties of EP panel and 70%EHS/EP2 phase change panel were tested and the results showed 70%EHS/EP2 phase change panel could remarkably decrease the maximum temperature during the melting process and obviously prolong the time of heat preservation in the cooling process which contributed to its good thermal control performance.

PCM has been investigated for solar energy storage in recent years. The magnesium nitrate hexahydrate was chosen as PCM due to the suitable melting temperature and high latent heat for solar thermal energy in this paper. In order to prevent the leakage during the phase transition, a novel form-stable composite PCM based on diatomite was prepared by the vacuum impregnation method. The micromorphology, chemical structure, crystalloid phase, thermal properties, supercooling degree, thermal conductivity and thermal reliability were investigated by SEM, XRD, FT-IR, DSC, self-designed device and hot-wire method. FT-IR spectra indicated that there was no chemical reaction during the preparation process. The supercooling tested by the self-designed device was just 0.3 °C which can be neglected. The melting and freezing temperature measured by DSC and step-cooling curve method were 88.89 °C and 83.5 °C, respectively. Furthermore, the latent heat was 69.39 J/g and the adsorptive capacity was 46.78% according the pure PCM used in this study. The thermal reliability test was performed and the results showed that the thermal properties basically unchanged after 2000 thermal cycles.

DNA methylation is a major mode of epigenetic regulation in the mammalian genome and is essential for embryonic development. The three catalytic DNA methyltransferases (Dnmts), Dnmt1, Dnmt3a, and Dnmt3b, catalyze the methylation of cytosine. Dnmt3b is highly expressed in chondrocytes and global knockout of Dnmt3b led to skeletal deformations and embryonic lethality, suggesting an essential role of Dnmt3b in endochondral bone formation. To further define the role of Dnmt3b in skeletal development, Dnmt3b was deleted in Col2 positive chondrocyte lineage cells. Both axial and appendicular skeletal size were reduced and bone mineralization delayed in Col2Cre(+);Dnmt3b(f/f) (Dnmt3b(Col2)) mice at E14.5 and E18.5. While Alcian Blue Hematoxylin/Orange G (ABH/OG) staining showed normal chondrocyte columns in control growth plates, the length of hypertrophic chondrocyte zone and type X collagen expression were decreased in E18.5 growth plates from Dnmt3b(Col2) mice. TUNEL and PCNA staining demonstrated that the delay in chondrocyte maturation observed in the Dnmt3b(Col2) growth plates was not secondary to altered chondrocyte apoptosis or proliferation. Complementary in vitro experiments were performed on primary sternal chondrocytes isolated from control and Dnmt3b(Col2) mice. Gene expression studies confirmed delayed terminal maturation as Mmp13 and Col10a1 expression was down-regulated in Dnmt3b(Col2) chondrocytes. In addition, alkaline phosphatase (ALP) and Alizarin Red staining confirmed that Dnmt3b deletion in chondrocytes delays in vitro chondrocyte hypertrophic differentiation and matrix mineralization. Mechanistically, Dnmt3b gene deletion resulted in decreased BMP signaling through reduction of Smad1 phosphorylation. These findings show that epigenetic factor, Dnmt3b is necessary for normal chondrocyte hypertrophic maturation and limb development.

We investigated enhancement of articular cartilage regeneration using a combination of human adipose derived stem cells (hADSCs) and TGF-β3 microspheres (MS) in vivo. Poly-lactic-co-glycolic acid (PLGA)MS were prepared using a solid/oil/water emulsion solvent evaporation-extraction method. The morphology of the MS was evaluated by scanning electron microscopy (SEM). The release characteristic of the TGF-β3 MS was evaluated. A New Zealand rabbit model for experimental osteoarthritis (OA) was established using the anterior medial meniscus excision method. Thirty OA rabbits were divided randomly into three groups according to different treatments of the right knee joints on day 7 after surgery: hADSCs/MS group received injection of both hADSCs and TGF-β3 MS; hADSCs group was injected with hADSCs; control group was injected with normal saline. Gross observation, histological staining and RT-PCR for collagen II and aggrecan) were used to assess the severity of OA and for evaluating the effect of combined use of hADSCs and TGF-β3 MS on articular cartilage regeneration in vivo. The MS were spherical with a smooth surface and the average diameter was 28 ± 2.3 µm. The encapsulation efficiency test showed that 73.8 ± 2.9% of TGF-β3 were encapsulated in the MS. The release of TGF- β3 lasted for at least 30 days. At both 6 and 12 weeks after injection, three groups exhibited different degrees of OA. Histological analysis showed that the hADSCs/MS group exhibited less OA than the hADSCs group, and the control group exhibited the most severe OA. Real-time RT-PCR showed that the gene expression of both collagen II and aggrecan were significantly up-regulated in the hADSCs/MS group. At 12 weeks after injection, the hADSCs/MS group also exhibited less OA than the other two groups. Combined use of hADSCs and TGF-β3 MS promoted articular cartilage regeneration in rabbit OA models.

Aims Transforming growth factor-β1 (TGF-β1) is a chondrogenic factor and has been reported to be able to enhance chondrocyte differentiation from bone marrow mesenchymal stem cells (BMSCs). Here we investigate the molecular mechanism through which TGF-β1 chronically promotes the repair of cartilage defect and inhibit chondrocyte hypertrophy. Main methods Animal models of full thickness cartilage defects were divided into three groups: model group, BMSCs group (treated with BMSCs/calcium alginate gel) and BMSCs + TGF-β1 group (treated with Lentivirus-TGF-β1-EGFP transduced BMSCs/calcium alginate gel). 4 and 8 weeks after treatment, macroscopic observation, histopathological study and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were done to analyze phenotypes of the animals. BMSCs were transduced with Lentivirus-TGF-β1-EGFP in vitro and Western blot analysis was performed. Key findings We found that TGF-β1-expressiing BMSCs improved the repair of the cartilage defect. The impaired cartilage contained higher amount of GAG and type II collagen and was integrated to the surrounding normal cartilage and higher content of GAG and type II collagen. The major events include increased expression of type II collagen following Smad2/3 phosphorylation, and inhibition of cartilage hypertrophy by increasing Yes-associated protein-1 (YAP-1) and inhibiting Runx2 and Col10 after the completion of chondrogenic differentiation. Significance We conclude that TGF-β1 is beneficial to chondrogenic differentiation of BMSCs via canonical Smad pathway to promote early-repairing of cartilage defect. Furthermore, TGF-β1 inhibits chondrocyte hypertrophy by decreasing hypertrophy marker gene expression via Hippo signaling. Long-term rational use of TGF-β1 may be an alternative approach in clinic for cartilage repair and regeneration.