SlPHL1 positively modulates acid phosphatase in response to phosphate starvation by directly activating the genes SlPAP10b and SlPAP15 in tomato.

Increased acid phosphatase (APase) activity is a prominent feature of tomato (Solanum lycopersicum) responses to inorganic phosphate (Pi) restriction. SlPHL1, a phosphate starvation response (PHR) transcription factor, has been identified as a positive regulator of low Pi (LP)-induced APase activity in tomato. However, the molecular mechanism underlying this regulation remains to be elucidated. Here, SlPHL1 was found to positively regulate the LP-induced expression of five potential purple acid phosphatase (PAP) genes, namely SlPAP7, SlPAP10b, SlPAP12, SlPAP15, and SlPAP17b. Furthermore, we provide evidence that SlPHL1 can stimulate transcription of these five genes by binding directly to the PHR1 binding sequence (P1BS) located on their promoters. The P1BS mutation notably weakened SlPHL1 binding to the promoters of SlPAP7, SlPAP12, and SlPAP17b but almost completely abolished SlPHL1 binding to the promoters of SlPAP10b and SlPAP15. As a result, the transcriptional activation of SlPHL1 on SlPAP10b and SlPAP15 was substantially diminished. In addition, not only did transient overexpression of either SlPAP10b or SlPAP15 in tobacco leaves increase APase activity, but overexpression of SlPAP15 in Arabidopsis and tomato also increased APase activity and promoted plant growth. Subsequently, two SPX proteins, SlSPX1 and SlSPX4, were shown to physically interact with SlPHL1. Moreover, SlSPX1 inhibited the transcriptional activation of SlPHL1 on SlPAP10b and SlPAP15 and negatively regulated the activity of APase. Taken together, these results demonstrate that SlPHL1-mediated LP signaling promotes APase activity by activating the transcription of SlPAP10b and SlPAP15, which may provide valuable insights into the mechanisms of tomato response to Pi-limited stress.

Using sequential bone cutting to titrate soft tissue balance in total knee arthroplasty effectively minimizes soft tissue release.

BACKGROUND: Achieving soft tissue balance while maintaining limb alignment within acceptable boundaries is crucial for successful total knee arthroplasty (TKA). We proposed a sequential bone cutting (SBC) technique to titrate the soft tissue balance in robot-assisted TKA to achieve the desired balance with minimum soft tissue release. METHODS: In total, 106 robot-assisted TKAs using the SBC technique were included. The preoperative hip-knee-ankle angle (HKA) was < 10° in 76 and ≥ 10° in 30 knees. The gaps were initially balanced with the help of the pre-resection balancing provided by the robotic system. Soft tissue balance and alignment were quantitatively measured after the initial bone cutting and final bone cutting. Additional adjustments (bone recuts and soft tissue releases) required to address soft tissue imbalance after initial bone cutting were recorded. The frequencies of soft tissue releases, soft tissue balance, and resultant alignment ≤ 3° were compared between non-severe (HKA < 10°) and severe deformity (HKA ≥ 10°) groups. RESULTS: Soft tissue balance was achieved in 45 knees (42.5%) after initial bone cutting and in 93 knees (87.7%) after final balancing. The postoperative alignment was within 3° from neutral in 87 knees (82.1%) and 3-5° in 17 knees (16.0%). For unbalanced knees (n = 61) after initial bone cutting, soft tissue release was avoided by SBC in 37 knees (60.7%) and was deemed necessary in 24 knees (39.3%). Soft tissue release was more likely to be avoided in the non-severe deformity cohort (86.8% [33 of 38]) than in the severe deformity cohort (17.4% [4 of 23]; p < 0.001). The non-severe deformity cohort showed a significantly higher rate of resultant alignment ≤ 3° from neutral than the severe deformity cohort (90.8% vs. 60.0%; p < 0.001). CONCLUSION: Pre-resection balancing is inappropriate to ensure soft tissue balance. The SBC technique is effective in minimizing soft tissue release while maintaining overall alignment within acceptable boundaries.

Hydrogel composite scaffolds achieve recruitment and chondrogenesis in cartilage tissue engineering applications.

BACKGROUND: The regeneration and repair of articular cartilage remains a major challenge for clinicians and scientists due to the poor intrinsic healing of this tissue. Since cartilage injuries are often clinically irregular, tissue-engineered scaffolds that can be easily molded to fill cartilage defects of any shape that fit tightly into the host cartilage are needed. METHOD: In this study, bone marrow mesenchymal stem cell (BMSC) affinity peptide sequence PFSSTKT (PFS)-modified chondrocyte extracellular matrix (ECM) particles combined with GelMA hydrogel were constructed. RESULTS: In vitro experiments showed that the pore size and porosity of the solid-supported composite scaffolds were appropriate and that the scaffolds provided a three-dimensional microenvironment supporting cell adhesion, proliferation and chondrogenic differentiation. In vitro experiments also showed that GelMA/ECM-PFS could regulate the migration of rabbit BMSCs. Two weeks after implantation in vivo, the GelMA/ECM-PFS functional scaffold system promoted the recruitment of endogenous mesenchymal stem cells from the defect site. GelMA/ECM-PFS achieved successful hyaline cartilage repair in rabbits in vivo, while the control treatment mostly resulted in fibrous tissue repair. CONCLUSION: This combination of endogenous cell recruitment and chondrogenesis is an ideal strategy for repairing irregular cartilage defects.

Development of Certified Reference Materials for the Determination of Apparent Amylose Content in Rice.

Apparent amylose content (AAC) is one of the most important parameters in rice quality evaluation. In this study, four rice reference materials used to test rice AAC were developed. The AAC of rice reference materials were measured by a spectrophotometric method with a defatting procedure, calibrated from potato amylose and waxy rice amylopectin at the absorption wavelengths of 620 and 720 nm. Homogeneity test (n = 20) was judged by F-test based on the mean squares of among and within bottles, and short- and long-term stability monitoring was performed by T-test to check if there was significant degradation at the delivery temperature of under 40 °C (14 days) and at 0-4 °C storage condition (18 months), respectively. After joint evaluation by ten laboratories, Dixion and Cochran statistical analyses were presented. The expanded uncertainties were calculated based on the uncertainty of homogeneity, short- and long-term stability, and inter-laboratory validation containing factor k = 2. It found that the four reference materials were homogenous and stable, and had the AAC (g/100 g, k = 2) of 2.96 ± 1.01, 10.68 ± 0.66, 17.18 ± 1.04, and 16.09 ± 1.29, respectively, at 620 nm, and 1.46 ± 0.49, 10.44 ± 0.56, 16.82 ± 0.75, and 24.33 ± 0.52, respectively, at 720 nm. It was indicated that 720 nm was more suitable for the determination of rice AAC with lower uncertainties. The determinations of the AAC of 11 rice varieties were carried out by two methods, the method without defatting and with calibration from the four rice reference materials and the method with a defatting procedure and calibrating from potato amylose and waxy rice amylopectin. It confirmed that the undefatted rice reference materials could achieve satisfactory results to test the rice samples with the AAC ranging from 1 to 25 g/100 g. It would greatly reduce the time cost and improve testing efficiency and applicability, and provide technical support for the high-quality development of the rice industry.

NRT2.1, a major contributor to cadmium uptake controlled by high-affinity nitrate transporters.

Management of nitrogen fertilizer is a good strategy for controlling cadmium (Cd) accumulation in plants. Some progress has already been made but much remains to be done. Here, we show that mutants with loss of function of nitrate transporter1.1 (NRT1.1) or nitrate transporter2.1 (NRT2.1) had lower Cd concentrations than wild-type plants under low-nitrate conditions. However, this was eliminated when plants were cultivated in nitrate-free medium or supplied with Cd and nitrate alternately. These findings indicate that inhibition of NRT1.1 or NRT2.1 activity reduces Cd accumulation in plants, and depends on the presence of nitrate. The results showing that nrt2.1-2 mutants had the lowest Cd concentrations compared with Col-0, nrt1.1 and nrt2.4 plants, proves that NRT2.1 is the major contributor to Cd uptake controlled by nitrate high-affinity transporters. NRT2.1 acts as the major contributor to nitrate uptake under Cd stress in low-nitrate conditions, and contributes about 50% to nitrate uptake, while NRT1.1 contributes only 10%, and little is known regarding the role of NRT2.2 and NRT2.4 on nitrate uptake in medium with 200 µM nitrate. Positive correlations between nitrate uptake and Cd concentration in plants were also observed. Collectively, NRT2.1 acts as the major contributor to Cd uptake by controlling nitrate uptake in nitrate high-affinity systems.

Chemistry-specific responses due to rice-microbe interactions in the rhizosphere to counteract mefenacet stress.

The widespread use of herbicides has raised considerable concern with regard to their harmful consequences on plant growth, crop yield and the soil ecological environment. It has been well documented that colonization of rhizobacteria in the plant root system has a positive effect on activation of plant defenses to protect the plant from damage. Using the platform of high-throughput analysis with tandem mass spectrometry and Illumina sequencing, we identified the specific activated rhizobacteria, the key growth stimulating substances and the metabolic pathways involved in seedling stage tolerance to mefenacet stress in rice. The relative abundance of beneficial rhizospheremicrobes such as Acidobacteria and Firmicutes increased with mefenacet treatment, indicating that the rhizosphere recruited some beneficial microbes to resist mefenacet stress. Mefenacet treatment induced alterations in several interlinked metabolic pathways, many of which were related to activation of defense response signaling, especially the indole-3-pyruvate pathway. Indole-3-acetaldehyde and indole-3-ethanol from this pathway may act as flexible storage pools for indole-3-acetic acid (IAA). Our findings also suggest that a significant increase of IAA produced by the enrichment of beneficial rhizospheremicrobes, for example genus Bacillus, alleviated the dwarfing phenomenon observed in hydroponic medium following mefenacet exposure, which may be a key signaling molecule primarily for phytostimulation and phytotolerance in microbe-plant interactions.

Insight of low-abundance proteins in rice leaves under Cd stress using combinatorial peptide ligand library technology.

Low-abundance proteins (LAPs) play a very important role in interaction, regulation, and metabolism of plant biological processes. A combinatorial peptide ligand library (CPLL) can solve the problem of high-abundance proteins (HAPs) masking LAPs and enlarging the dynamic range of protein concentrations perfectly and be considered as one of the most advanced approaches for plant proteomics research. In this paper, a proper CPLL method to rice leaf proteins was established for the first time and 1056 proteins were identified in rice leaf extracts, and 624 (59.1%) LAPs were newly detected after CPLL. Based on this technology, we detected the response of rice to Cd stress and analyzed the differential LAPs and the biological significance of misexpressed proteins before and after Cd stress by bioinformatics analysis. An important contribution has also been made to a better understanding of the complex mechanisms by which rice adapts to Cd stress. Graphical abstract.

Water management affects arsenic uptake and translocation by regulating arsenic bioavailability, transporter expression and thiol metabolism in rice (Oryza sativa L.).

Water management is an economic and effective strategy to reduce arsenic (As) accumulation in rice grains, but little is known about the effect of water management on the migration and transformation of As in the soil-rice system. In this study, the effect of the continually (CF) and intermittent flooding (IF) treatments on the dynamic change of As in the rhizosphere soil-pore water-iron plaque-rice system was systematically investigated using pot experiments. The expressions of genes involved in As uptake and translocation in rice plants under different water management treatments were further examined. Results showed that the total As concentration in brown rice was increased by 50.8% in the CF treatment compared to the IF treatment, and dimethylarsinic acid (DMA) made greater contribution (from 15.5% to 29.2%) to total As increase in brown rice under the CF treatment. The CF treatment increased As bioavailability in the rhizosphere soil and soil pore water, which enhanced As uptake and transport to the xylem in rice plants by inducing the expressions of silicon transporter genes (OsLsi1 and OsLsi2) compared to the IF treatment. Moreover, the CF treatment increased As translocation from roots to shoots by reducing soil available sulfur and phytochelatins (PCs) biosynthesis and vacuolar sequestration in rice roots compared with the IF treatment. The study provides insight into the physiological and molecular mechanisms underlying As uptake and translocation in rice plants under different water regimes, which will be helpful for adopting the irrigation technique to mitigate excessive As accumulation in rice grains and associated health risk to humans.

Simultaneous determination of three organotin pesticides in fruits and vegetables by high-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: The presence of organotins in the environment affects food safety, making it important to monitor the levels of organotin pesticides (OTPs) in fruit and vegetable samples. METHODS: In the present study, a simple and low cost method for simultaneous determination of three OTPs (azocyclotin, fenbutatin oxide and triphenyltin hydroxide) in vegetable and fruit samples was developed and validated, based on solid-phase extraction and liquid chromatography/tandem mass spectrometry. RESULTS: Extraction with acetonitrile containing 0.2% formic acid positively affected the recoveries of the three OTPs. Moreover, the simultaneous purification of the three OTPs was the most efficient using mixed-mode cation-exchange cartridges and 5.0% ammonium hydroxide in methanol as eluent, and, in this case, mild matrix effects (-9.3% to 21.6%) were obtained for the three OTPs monitored. The developed method reached limits of quantification of 1 µg kg-1 , and linearity was satisfactory, with correlation coefficients >0.995. A fortification study showed that when spiked at 1.0-50.0 µg kg-1 the mean trueness values were from 72.3 to 110.0% in all matrices (three vegetables and three fruits). The intra-day precision was <14.1%, and the inter-day precision (n = 11) was <18.2%. CONCLUSIONS: The proposed method was successfully applied to the simultaneous analysis of three OTPs in vegetables and fruits.

Dissipation of two field-incurred pesticides and three degradation products in rice (Oryza sativa L.) from harvest to dining table.

BACKGROUND: High levels of harmful pesticide residues in rice can cause undesirable side effects and are a source of great concern to consumers. Reduction of pesticide residues to provide rice security has thus became an urgent problem. RESULTS: In this study, the effects of commercial and home processing on removal of chlorpyrifos and carbosulfan residues from rice, and the formation of metabolites during processing, were studied. The results showed that 3,5,6-trichloro-2-pyridinol (0.87 mg kg-1 ) and carbofuran (0.43 mg kg-1 ) were the predominant components detected in paddy rice. All detected residues were primarily deposited on the rice hull and bran. Washing twice followed by high-pressure cooking was able to further decrease residues in polished rice with the processing factor value <0.25. Following application of pesticides at the recommended rate and twice the recommended rate, with a preharvest interval of 28 days, changes in residues from harvest to dining table based on efficient processing techniques were investigated. The final residues dropped to below maximum residue levels after washing twice followed by high-pressure cooking. CONCLUSION: This simple cooking process thus reduces the risk of dietary exposure, and it is recommended that it is adopted by all consumers. © 2019 Society of Chemical Industry.

Co-culture systems-based strategies for articular cartilage tissue engineering.

Cartilage engineering facilitates repair and regeneration of damaged cartilage using engineered tissue that restores the functional properties of the impaired joint. The seed cells used most frequently in tissue engineering, are chondrocytes and mesenchymal stem cells. Seed cells activity plays a key role in the regeneration of functional cartilage tissue. However, seed cells undergo undesirable changes after in vitro processing procedures, such as degeneration of cartilage cells and induced hypertrophy of mesenchymal stem cells, which hinder cartilage tissue engineering. Compared to monoculture, which does not mimic the in vivo cellular environment, co-culture technology provides a more realistic microenvironment in terms of various physical, chemical, and biological factors. Co-culture technology is used in cartilage tissue engineering to overcome obstacles related to the degeneration of seed cells, and shows promise for cartilage regeneration and repair. In this review, we focus first on existing co-culture systems for cartilage tissue engineering and related fields, and discuss the conditions and mechanisms thereof. This is followed by methods for optimizing seed cell co-culture conditions to generate functional neo-cartilage tissue, which will lead to a new era in cartilage tissue engineering.

Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem Cells: Characterization, Differentiation, and Applications in Cartilage Tissue Engineering.

Articular cartilage defects have very limited self-repair potential, and traditional bone marrow-stimulating therapy is not effective. Cartilage tissue engineering using bone marrow mesenchymal stem cells (BMSCs) and adipose tissue-derived mesenchymal stem cells (ADSCs) is considered an attractive treatment for cartilage lesions and osteoarthritis. However, studies proved that both BMSCs and ADSCs have their own advantages and shortcomings, including their sources, isolation methods, characterizations and differentiation potential. Understanding the properties and differences between ADSCs and BMSCs is important for clinical application in cartilage regeneration. This review provides an overview of BMSCs and ADSCs based on their characterization, isolation. Then, we summarized their differentiation potential in different experimental conditions. Finally, we discuss the applications of BMSCs and ADSCs in scaffold-free and scaffold-based cartilage tissue engineering. Based on different properties of BMSCs and ADSCs, and patient's physical condition, a more suitable therapeutic strategy can be selected.

Native tissue-based strategies for meniscus repair and regeneration.

Meniscus injuries appear to be becoming increasingly common and pose a challenge for orthopedic surgeons. However, there is no curative approach for dealing with defects in the inner meniscus region due to its avascular nature. Numerous strategies have been applied to regenerate and repair meniscus defects and native tissue-based strategies have received much attention. Native tissue usually has good biocompatibility, excellent mechanical properties and a suitable microenvironment for cellular growth, adhesion, redifferentiation, extracellular matrix deposition and remodeling. Classically, native tissue-based strategies for meniscus repair and regeneration are divided into autogenous and heterogeneous tissue transplantation. Autogenous tissue transplantation is performed more widely than heterogeneous tissue transplantation because there is no immunological rejection and the success rates are higher. This review first discusses the native meniscus structure and function and then focuses on the use of the autogenous tissue for meniscus repair and regeneration. Finally, it summarizes the advantages and disadvantages of heterogeneous tissue transplantation. We hope that this review provides some suggestions for the future design of meniscus repair and regeneration strategies.

Separation of selenium species and their sensitive determination in rice samples by ion-pairing reversed-phase liquid chromatography with inductively coupled plasma tandem mass spectrometry.

A highly sensitive method was developed for the simultaneous separation and determination of organic and inorganic selenium species in rice by ion-pairing reversed-phase chromatography combined with inductively coupled plasma tandem mass spectrometry. To achieve a good separation of these species, a comparison between anion-exchange chromatography and ion-pairing reversed-phase chromatography was performed. The results indicated that ion-pairing reversed-phase chromatography was more suitable due to better separation and higher sensitivity for all analytes. In this case, a StableBond C18 column proved to be more robust or to have a better resolution than other C18 columns, when 0.5 mM tetrabutylammonium hydroxide and 10 mM ammonium acetate at pH 5.5 were used as the mobile phase. Moreover, an excellent sensitivity was obtained in terms of interferences by means of tandem mass spectrometry in the hydrogen mode. The detection limits were 0.02-0.12 µg/L, and recoveries of five selenium species were 75-114%, with relative standard deviations ≤ 9.4%. This method was successfully applied to the analysis of rice samples. Compared with previous studies, the proposed method not only gave comparable results when used for measuring selenium-enriched rice, but it can provide greater sensitivity for the detection of low concentrations of selenium species in rice.

Isolation, identification, and characterization of Ustilaginoidea virens from rice false smut balls with high ustilotoxin production potential.

Ustilaginoidea (U.) virens grows on rice grains and leads to significant rice yield losses in most of the major rice producing areas. Meanwhile, ustiloxins produced by U. virens are a serious hazard to human health and ecological safety of farmlands. The other key point is that ustiloxins have been regarded as a novel resource with their potential in the treatment of cancers. There is no better way to extract ustiloxins than from pure culture of the high ustilotoxin-producing strains. U. virens has become a key research organism. However, due to the presence of some interference components, it is a certain difficulty in the successful isolation of the strain from the false smut balls. We present here a detailed study based on the separation, screening and identification of high ustiloxins-producing strains of U. virens. Through this study, we got a satisfactory success rate of separation and provided a good solution to the problem of separation. At the same time, this study provides quality resources for researchers interested in ustiloxins as anticancer agents.

Characterization of the phytochelatins and their derivatives in rice exposed to cadmium based on high-performance liquid chromatography coupled with data-dependent hybrid linear ion trap orbitrap mass spectrometry.

RATIONALE: The identification and quantification of phytochelatins (PCs) and their derivatives are important to understand their roles in plant growth and development. A method couplling high-performance liquid chromatography with hybrid linear ion trap Orbitrap mass spectrometry (HPLC-LTQ/Orbitrap) was developed to screen PCs that have the same characteristic product ions. This approach was used for the fragmentation pattern analysis of glutathione (GSH) and PC standards, which allowed identification of the fragmentation pathways of their derivatives isolated from rice roots, stems and leaves. METHODS: In this study, we developed a method to detect and identify PCs and their derivatives in rice based on HPLC/LTQ-Orbitrap. Spectrum interpretation and MS/MS fragmentation patterns of PCs provide sufficient information to discover the novel PC derivatives. This approach includes precursor ion scan and product ion scan to detect and character the novel PC derivatives. RESULTS: Based on HCD-MS/MS fragmentation patterns, four PCs and 18 PC derivatives were identified. Among them, seven PC derivatives, i.e., iso-PC2 (Asn), iso-PC3 (Asn), iso-PC2 (Cys), des-γGlu-iso-PC3 (Ser), des-Cys-iso-PC2 (Glu), des-Cys-iso-PC3 (Glu) and des-Cys-iso-PC4 (Glu), have not been previously reported. This method was validated by profiling GSH, PCs and PC derivatives in rice. Preliminary results revealed that PCs and their derivatives, except GSH, are markedly induced by Cd treatment. CONCLUSIONS: The HPLC/LTQ-Orbitrap method was successfully developed for the identification of PCs and their derivatives. The C-terminal linked to Gly is replaced with Glu, Ser, Asn, Gln or Cys, thereby creating a family of chemicals that share several structural properties. This technique could be particularly useful for investigators studying plant metabolomics. Copyright © 2016 John Wiley & Sons, Ltd.

Robust method for the analysis of phytochelatins in rice by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry based on polymeric column materials.

A sensitive and robust high-performance liquid chromatography coupled with electrospray tandem mass spectrometry method for the identification and quantification of glutathione and phytochelatins from rice was developed. Homogenized samples were extracted with water containing 100 mM dithiothreitol, and solid-phase extraction using polymer anion exchange resin was employed for sample purification. Chromatography was performed on a polymeric column with acetonitrile and water containing 0.1% formic acid as the mobile phase at the flow rate of 300 µL/min. The limit of quantitation was 6-100 nM. This assay showed excellent linearity for both glutathione and phytochelatins over physiological normal ranges, with correlation coefficients (r) > 0.9976. Recoveries for four biothiols were within the range of 76-118%, within relative standard deviations less than 15%. The intraday precision (n = 7) was 2.1-13.3%, and the interday precision over 15 days was 4.3-15.2%. The optimized method was applied to analyze tissue samples from rice grown using nutrient solutions with three different cadmium concentrations (0, 50, and 100 µM). With increasing cadmium concentrations, the content of phytochelatin 2 and phytochelatin 3 in rice roots increased, in contrast to most phytochelatins, and the content of glutathione in rice stems and roots decreased significantly.

Mutation of OsNRAMP5 reduces cadmium xylem and phloem transport in rice plants and its physiological mechanism.

Natural resistance associated macrophage protein 5 (NRAMP5) is a key transporter for cadmium (Cd) uptake by rice roots; however, the effect of OsNRAMP5 on Cd translocation and redistribution in rice plants remains unknown. In this study, an extremely low Cd-accumulation mutant (lcd1) and wild type (WT) plants were utilized to investigate the effect of OsNRAMP5 mutation on Cd translocation and redistribution via the xylem and phloem and its possible physiological mechanism using field, hydroponic and isotope-labelling experiments. The results showed that OsNRAMP5 mutation reduced xylem and phloem transport of Cd, due to remarkably lower Cd translocation from roots to shoots and from the leaves Ⅰ-Ⅲ to their corresponding nodes, as well as lower Cd concentrations in xylem and phloem sap of lcd1 compared to WT plants. Mutation of OsNRAMP5 reduced Cd translocation from roots to shoots in lcd1 plants by increasing Cd deposition in cellulose of root cell walls and reducing OsHMA2-and OsCCX2-mediated xylem loading of Cd, and the citric acid- and tartaric acid-mediated long-distance xylem transport of Cd. Moreover, OsNRAMP5 mutation inhibited Cd redistribution from flag leaves to nodes and panicles in lcd1 plants by increasing Cd sequestration in cellulose and vacuoles, and decreasing OsLCT1-mediated Cd phloem transport in flag leaves.

Robotic-assisted differential total knee arthroplasty with patient-specific implants: surgical techniques and preliminary results.

OBJECTIVE: In total knee arthroplasty (TKA), achieving soft-tissue balance while retaining acceptable lower limb alignment is sometimes difficult and may lead to patient dissatisfaction. Theoretically, patient-specific implants can bring great benefits, while the lack of precise surgical tools may hinder the improvement of outcomes. The objective of this study was to illustrate surgical techniques and evaluate kinematics and early clinical outcomes of robotic-assisted TKA using patient-specific implants. METHODS: Based on preoperative CT scan, femoral and tibial components were 3D printed. Medial and lateral tibial liners were separate with different thicknesses, posterior slopes and conformity. TiRobot Recon Robot was used for surgery, and was armed with smart tools that quantify gap, force and femoral-tibial track. We collected data on demographics, intraoperative gap balance and femoral-tibial motion. In the follow-up, we evaluated the range of motion, Visual Analogue Scale (VAS), forgotten joint score (FJS), Knee injury and Osteoarthritis Outcome Score, Joint Replacement (KOOS, JR) score. Radiological data were also harvested. RESULTS: Fifteen patients (17 knees) were enrolled with a mean age of 64.6 ± 6.4 (53-76) years. In 5 knees, we used symmetric tibial liners, the rest were asymmetric. After surgery, the average alignment was 1.6 ± 2.0 (-3-5) degrees varus. The average follow-up lasted 6.7 ± 4.2 (1-14) months. The mean visual analogue scale was 0.8 ± 0.7 (0-2), FJS was 62.4 ± 25.3 (0-87), KOOS was 86.5 ± 9.4 (57-97). 11 patients were "very satisfied", 3 were "satisfied" with the result, and one patient was neutral due to restricted extension and unsatisfactory rehabilitation at five months' follow-up. CONCLUSIONS: With patient-specific implants and robotics, TKA could be performed by a mathematical way, which was dubbed a "differential" TKA. Intraoperative kinematics was excellent in terms of gap-force balancing and femoral-tibial relative motion. Preliminary clinical outcomes were overall satisfactory.

[Construction of a novel tissue engineered meniscus scaffold based on low temperature deposition three-dimenisonal printing technology].

Objective: To investigate the construction of a novel tissue engineered meniscus scaffold based on low temperature deposition three-dimenisonal (3D) printing technology and evaluate its biocompatibility. Methods: The fresh pig meniscus was decellularized by improved physicochemical method to obtain decellularized meniscus matrix homogenate. Gross observation, HE staining, and DAPI staining were used to observe the decellularization effect. Toluidine blue staining, safranin O staining, and sirius red staining were used to evaluate the retention of mucopolysaccharide and collagen. Then, the decellularized meniscus matrix bioink was prepared, and the new tissue engineered meniscus scaffold was prepared by low temperature deposition 3D printing technology. Scanning electron microscopy was used to observe the microstructure. After co-culture with adipose-derived stem cells, the cell compatibility of the scaffolds was observed by cell counting kit 8 (CCK-8), and the cell activity and morphology were observed by dead/live cell staining and cytoskeleton staining. The inflammatory cell infiltration and degradation of the scaffolds were evaluated by subcutaneous experiment in rats. Results: The decellularized meniscus matrix homogenate appeared as a transparent gel. DAPI and histological staining showed that the immunogenic nucleic acids were effectively removed and the active components of mucopolysaccharide and collagen were remained. The new tissue engineered meniscus scaffolds was constructed by low temperature deposition 3D printing technology and it had macroporous-microporous microstructures under scanning electron microscopy. CCK-8 test showed that the scaffolds had good cell compatibility. Dead/live cell staining showed that the scaffold could effectively maintain cell viability (>90%). Cytoskeleton staining showed that the scaffolds were benefit for cell adhesion and spreading. After 1 week of subcutaneous implantation of the scaffolds in rats, there was a mild inflammatory response, but no significant inflammatory response was observed after 3 weeks, and the scaffolds gradually degraded. Conclusion: The novel tissue engineered meniscus scaffold constructed by low temperature deposition 3D printing technology has a graded macroporous-microporous microstructure and good cytocompatibility, which is conducive to cell adhesion and growth, laying the foundation for the in vivo research of tissue engineered meniscus scaffolds in the next step.