Identification of Periostin Positive Cell Population during the Long-Term Culture of Mouse Tendon-Derived Cells in Late Passage.

Tendon-derived cells exhibit phenotypic changes and gradually lose their proliferative capacity during serial passages in vitro. This study aimed to characterize the changes in the growth and stem cell characteristics of tendon-derived cells over a long-term culture. Mouse flexor digitorum profundus tendon-derived cells were obtained by enzymatic digestion and seeded at an initial density of 5,000/cm2. Cells were characterized by morphology, growth, senescence staining, trilineage differentiation assays, real-time polymerase chain reaction, immunocytochemistry, flow cytometry, and RNA sequencing analysis. Tendon-derived cells underwent a proliferative stage in the first three passages, followed by a gradual senescence. However, a novel cell population expressing periostin (Postn+) emerged during the long-term culture from passages 5-8, which possessed a high rate of proliferation without significant senescence over successive passages. Compared to early passage cells, Postn+ cells exhibited enhanced osteogenic differentiation potential and attenuated chondrogenic differentiation potential, decreased expression of SSEA-1, Oct3/4, tenomodulin, scleraxis, CD90.2, CD73, CD105, Sca-1, and CD44, and increased expression of collagen III and CD34. RNA-sequencing analysis of Postn+ and early passage cells identified 908 differentially expressed genes, with extracellular matrix-receptor interaction and focal adhesion as the top pathways, and integrins as hub genes. This study highlights the dynamics of tendon-derived cells during serial passages. We identify a Postn+ cell population during long-term culture in late passages, with high proliferative ability and prominent osteogenic differentiation potential. Further investigations are needed to elucidate the origin and potential applications of Postn+ tendon-derived cells.

Gap Resistance and Tensile Strength of a Q Suture Technique During Curved Loading: An Ex Vivo Porcine Flexor Tendon Study.

PURPOSE: This study aimed to determine the mechanical properties of the double Q suture technique in angular motion and to compare the gap formation associated with tendon repairs during curved and linear loading. METHODS: Eighty porcine flexor tendons were repaired with one of two 4-strand sutures: double Q suture or double modified Kessler plus peripheral running sutures. The repaired tendons were cyclically loaded sequentially against a pulley with a radius of 2.0, 1.5, and 1.0 cm or linearly without any pulleys. The number of tendons that formed an initial or 2-mm gap at the repair site during cyclic loading, the gap size between tendon ends when cyclic loading ended, and the ultimate strength were recorded. RESULTS: The gap at the repair site formed gradually from the dorsal to volar aspect during curved loading. No double Q repairs, but half of the double Kessler plus running suture repairs, formed an initial or 2-mm gap on the volar aspect during curved loading. The double Q group had a significantly smaller gap size on the dorsal aspect than the double Kessler plus running suture group at all three radii of curvature. The ultimate strength was similar between the two groups. There were no significant differences in linear motion between these two repairs. CONCLUSIONS: The double Q suture is superior to the conventional 4-strand tendon core suture plus running peripheral sutures in gap resistance in angular motion. This study provides insight into the formation of an unbalanced gap on the dorsal and volar aspects of tendon repair during curved loading. CLINICAL RELEVANCE: The double Q suture provides a simple and efficient option for flexor tendon repair considering the high risk of gap formation on the dorsal aspects of the tendon repair in angular motion.

Cumulative Risk Assessment of Exposure to Heavy Metals through Aquatic Products in China.

OBJECTIVE: This study aimed to assess the risk of cumulative exposure to Pb, Cd, Hg, and iAs through aquatic products consumed by Chinese people. METHODS: Heavy metal concentration data were obtained from the national food contamination monitoring program during 2013-2017. Consumption data were derived from the China National Food Consumption Survey in 2014 and the relative potency factor (RPF) method was used to estimate cumulative exposure for neurotoxicity and nephrotoxicity. RESULTS: The results demonstrated that the cumulative exposure based on neurotoxicity was below the lower confidence limit on benchmark doses of lead (BMDL 01) for nephrotoxicity and the cumulative exposures were all lower than the provisional tolerable monthly intake (PTMI) of Cd. However, the margin of exposure values (MOEs) of the cumulative exposures for neurotoxicity in the 2-6 year-old group was close to 1 and the cumulative exposure level for nephrotoxicity accounted for 90.21 % of the PTMI. CONCLUSION: The cumulative exposures of the 2-6 year-old group to the four heavy metals did not reach (but came close to) the corresponding safety threshold for both neurotoxicity and nephrotoxicity. Given that there are still other food sources of these four heavy metals, it is necessary to more closely study their cumulative health effects.

The impact of transverse components on 4-strand tendon repairs.

We investigated the effects of the transverse components of a tendon core suture on tensile resistance and strength of 4-strand repairs. Forty-four pig flexor tendons were repaired with one of the following four methods: double Tsuge, U-shaped, 4-strand cross and 4-strand rectangular repairs. We recorded the number of the repaired tendons that formed a 2 mm gap between the tendon ends during cyclic loading for 20 cycles, stiffness of the tendon at the 1st and 20th cycle, gap distance at the repair site and ultimate strength of the repair at the 20th cycle. When transverse components were added to the core suture, a greater number of tendons formed a 2 mm gap during cyclic loading. The stiffness gradually decreased, and the repair site's gap distance after cyclic loading increased with the presence of transverse components of the sutures. We conclude that the core suture's transverse components negatively impact the tensile resistance of 4-strand tendon repairs.

Using Q Suture to Enhance Resistance to Gap Formation and Tensile Strength of Repaired Flexor Tendons.

Peripheral epitendinous sutures are believed to enhance core suture strength in tendon repair and decrease the risk of gapping between tendon ends. Here Q suture, an alternative to peripheral sutures, is presented for the use in tendon repair. Its effects on gap formation and tensile strength of the repaired tendons were compared with conventional running peripheral sutures. Three 2-strand sutures and three 4-strand sutures were used in repairing porcine tendons. The time required for performing 2Q and running sutures were recorded. The repaired tendons were subjected to a cyclic loading test, and the cycle number, during which a 2-mm gap was formed, was determined. After the cyclic loading, the gap size at the tendon ends and the ultimate strength of the repaired tendons were measured. Augmentation with the Q sutures reduced the number of tendons showing 2-mm gaps at tendon ends during cyclic loading. With addition of Q sutures 2-strand sutures significantly increased the ultimate strength of the repaired tendons and 4-strand sutures decreased the gap distance at the repair site of tendons. The time required for performing 2Q sutures was significantly less than that for running sutures. Therefore, we conclude that the Q suture is efficient in enhancing the tensile resistance and tendon repair strength and can be an alternative to conventional peripheral sutures.

Growth and Stem Cell Characteristics of Tendon-Derived Cells with Different Initial Seeding Densities: An In Vitro Study in Mouse Flexor Tendon Cells.

Tendon stem/progenitor cells (TSPCs) are considered promising seed cells for tendon regeneration. Previous studies reported that a low seeding density favors TSPC growth, whereas a high seeding density favors tenocyte growth. We aimed to distinguish TSPCs from tenocytes by seeding tendon-derived cells at a density gradient. In this study, tendon-derived cells were isolated from flexor digitorum profundus tendons of mice and seeded at the initial densities of 50, 500, 5,000, and 50,000/cm2. We found that distinct cell colonies were formed from cells with initial seeding densities of 50 and 500/cm2, but colonies were not discernible for cells seeded at 5,000 and 50,000/cm2. There was a positive correlation between cell proliferation rate and seeding density, but a negative correlation between cell senescence and seeding density. The cell proliferation rate decreased gradually during serial passages. All cells exhibited restricted differentiation potentials, and expressed stem cell markers and relatively high levels of tenogenic markers without notable differences among cells seeded at different densities. We concluded that a pure population of TSPCs could not be isolated from mouse digital flexor tendons through culturing cells at a density gradient. Cells seeded at low densities had very limited proliferative ability and did not show more prominent stem cell characteristics when compared with cells seeded at high densities.

Transcriptome profiling of digital flexor tendons after injury in a chicken model.

BACKGROUND: Modulation of tendon healing remains a challenge because of our limited understanding of the tendon repair process. Therefore, we performed the present study to provide a global perspective of the gene expression profiles of tendons after injury and identify the molecular signals driving the tendon repair process. RESULTS: The gene expression profiles of flexor digitorum profundus tendons in a chicken model were assayed on day 3, weeks 1, 2, 4, and 6 after injury using the Affymetrix microarray system. Principal component analysis (PCA) and hierarchical cluster analysis of the differentially expressed genes showed three distinct clusters corresponding to different phases of the tendon healing period. Gene ontology (GO) analysis identified regulation of cell proliferation and cell adhesion as the most enriched biological processes. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis revealed that the cytokine-cytokine receptor interaction and extracellular matrix (ECM)-receptor interaction pathways were the most impacted. Weighted gene co-expression network analysis (WGCNA) demonstrated four distinct patterns of gene expressions during tendon healing. Cell adhesion and ECM activities were mainly associated with genes with drastic increase in expression 6 weeks after injury. The protein-protein interaction (PPI) networks were constructed to identify the key signaling pathways and hub genes involved. CONCLUSIONS: The comprehensive analysis of the biological functions and interactions of the genes differentially expressed during tendon healing provides a valuable resource to understand the molecular mechanisms underlying tendon healing and to predict regulatory targets for the genetic engineering of tendon repair. Tendon healing, Adhesion, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Weighted Gene Co-expression Network Analysis, Protein-protein Interaction.

The Effects of Hypoxia-Reoxygenation in Mouse Digital Flexor Tendon-Derived Cells.

OBJECTIVE: Ischemia-reperfusion injury refers to the exacerbated and irreversible tissue damage caused by blood flow restoration after a period of ischemia. The hypoxia-reoxygenation (H/R) model in vitro is ideal for studying ischemia-reperfusion injury at the cellular level. We employed this model and investigated the effects of cobalt chloride- (CoCl2-) induced H/R in cells derived from mouse digital flexor tendons. MATERIALS AND METHODS: Various H/R conditions were simulated via treatment of tendon-derived cells with different concentrations of CoCl2 for 24 h, followed by removal of CoCl2 to restore a normal oxygen state for up to 96 h. Cell viability was measured using the Cell Counting Kit-8 (CCK-8) assay. Cell growth was determined via observation of cell morphology and proliferation. Oxidative stress markers and mitochondrial activity were detected. The expression levels of hypoxia-inducible factor- (HIF-) 1α, vascular endothelial growth factor-A (VEGF-A), collagen I, and collagen III were determined using Western blot (WB), real-time PCR, and immunofluorescence staining. Cellular apoptosis was analyzed via flow cytometry, and the expression of apoptosis-related proteins Bax and bcl-2 was examined using WB. RESULTS: The cells treated with low concentrations of CoCl2 showed significantly increased cell viability after reoxygenation. The increase in cell viability was even more pronounced in cells that had been treated with high concentrations of CoCl2. Under H/R conditions, cell morphology and growth were unchanged, while oxidative stress reaction was induced and mitochondrial activity was increased. H/R exerted opposite effects on the expression of HIF-1α mRNA and protein. Meanwhile, the expression of VEGF-A was upregulated, whereas collagen type I and type III were significantly downregulated. The level of cellular apoptosis did not show significant changes during H/R, despite the significantly increased Bax protein and reduced bcl-2 protein levels that led to an increase in the Bax/bcl-2 ratio during reoxygenation. CONCLUSIONS: Tendon-derived cells were highly tolerant to the hypoxic environments induced by CoCl2. Reoxygenation after hypoxia preconditioning promoted cell viability, especially in cells treated with high concentrations of CoCl2. H/R conditions caused oxidative stress responses but did not affect cell growth. The H/R process had a notable impact on collagen production and expression of apoptosis-related proteins by tendon-derived cells, while the level of cellular apoptosis remained unchanged.

Effects of a Q Suture Technique on Resistance to Gap Formation and Tensile Strength of Repaired Tendons: An Ex Vivo Mechanical Study.

PURPOSE: The repair of digital flexor tendons following laceration should aim to prevent gapping at the repair site and restore the tensile strength of the tendons to facilitate postoperative movement. We present here a simple Q suture and test its effects on gap formation and tensile strength of the repaired tendons. METHODS: Sixty porcine tendons were repaired with 3 2-strand sutures (Kessler, Kessler plus 2Q, and Kessler plus running sutures) and 3 4-strand sutures (double Kessler, double Kessler plus 2Q, and double Kessler plus running sutures). The specimens were subjected to a cyclic loading. At each cycle, the number of tendons that initiated gapping or formed a 2-mm gap at the repair site was determined. After the cyclic load testing, the gap distance between tendon ends and the ultimate strength of the repaired tendons was measured. RESULTS: In both 2-strand and 4-strand tendon repairs, augmentation by insertion of the 2Q sutures reduced the number of tendons that showed 2-mm gaps ends during loading. Compared with the single Kessler and Kessler plus running sutures, Kessler plus 2Q suture significantly increased the ultimate strength of the tendon repair. Compared with the double Kessler and double Kessler plus running sutures, double Kessler plus 2Q suture significantly decreased the gap distance at the repair site after cyclic loading. CONCLUSIONS: The Q suture technique effectively enhances the resistance to gap formation of 2-strand and 4-stand tendon repair. It also improves the tensile strength of 2-strand Kessler repairs. CLINICAL RELEVANCE: The Q suture is a simple technique that can resist gap formation and strengthen the tensile strength of the repaired tendons in the laboratory setting.

Trans Fatty Acid Levels in Foods and Intakes among Population Aged 3 Years and above in Beijing and Guangzhou Cities, China.

OBJECTIVE: To understand the dietary intake levels of trans fatty acids (TFA) in a Chinese population and establish a basis for health risk assessment of trans fatty acids. METHODS: The TFA contents data of 2613 food items and food consumption data of 10,533 people aged 3 years and above in two large cities in China were matched and a simple assessment method was used to estimate the distribution of dietary TFA intake. RESULTS: The mean content of TFA was highest in margarine (1.68 ± 0.83 g/100g), followed by chocolate and candy (0.89 ± 2.68 g/100g), edible vegetable oils (0.86 ± 0.82 g/100g), milk (0.83 ± 1.56 g/100g), and bakery foods (0.41 ± 0.91 g/100g). TFA intake accounted for 0.34%, 0.30%, 0.32%, and 0.29% of the total energy intake in the 3-6, 7-12, 13-17, and ⋝18 year age groups, respectively. Of the populations studied, 0.42% demonstrated TFA intakes (as percentage of energy intake) greater than 1%. The main sources of dietary TFA intake were edible vegetable oils, milk, mutton, and beef, and baked foods, which accounted for 49.8%, 16.56%, 12.21%, and 8.87%, respectively. CONCLUSION: The current intake of TFA among people in two cities did not appear to be of major health concern regarding the threshold of TFA intake as the percentage of total energy recommended by the World Health Organization. Because most TFA were derived from industrially processed foods, the government should reinforce nutrition labeling and regulate food producers to further reduce TFA in food and to provide scientific instruction for consumers to make sound choices.

The important roles of RET, VEGFR2 and the RAF/MEK/ERK pathway in cancer treatment with sorafenib.

AIM: To elucidate the roles of receptor tyrosine kinases RET and VEGFR2 and the RAF/MEK/ERK signaling cascade in cancer treatment with sorafenib. METHODS: The cell lines A549, HeLa, and HepG2 were tested. The enzyme activity was examined under cell-free conditions using 384-well microplate assays. Cell proliferation was evaluated using the Invitrogen Alarmar Blue assay. Gene expression was analyzed using the Invitrogen SYBR Green expression assays with a sequence detection system. Protein expression analysis was performed using Western blotting. RESULTS: Sorafenib potently suppressed the activities of cRAF, VEGFR2, and RET with IC(50) values of 20.9, 4 and 0.4 nmol/L, respectively. Sorafenib inhibited cRAF, VEGFR2, and RET via non-ATP-competitive, ATP-competitive and mixed-type modes, respectively. In contrast, sorafenib exerted only moderate cytotoxic effects on the proliferation of the 3 cell lines. The IC(50) values for inhibition of A549, HeLa, and HepG2 cells were 8572, 4163, and 8338 nmol/L, respectively. In the 3 cell lines, sorafenib suppressed the cell proliferation mainly by blocking the MEK/ERK downstream pathway at the posttranscriptional level, which in turn regulated related gene expression via a feed-back mechanism. CONCLUSION: This study provides novel evidence that protein kinases RET and VEGFR2 play crucial roles in cancer treatment with sorafenib.

Cellular apoptosis and proliferation in the middle and late intrasynovial tendon healing periods.

PURPOSE: Cellular apoptosis might be an important molecular event in the middle or late healing periods of intrasynovial tendons, but this has not been studied. We aimed to investigate cellular apoptosis and corresponding cellular proliferation in the middle and late healing stages of intrasynovial tendons. METHODS: The flexor digitorum profundus tendons of 48 long toes (24 chickens) were completely transected within the sheath region and were repaired surgically. At days 28, 42, 56, and 84 after surgery, tendons were harvested and sectioned. In situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to detect apoptotic cells. The sections were stained immunofluorescently with antibodies to proliferating cell nuclear antigen to assess proliferation and to Bcl-2 (an anti-apoptotic protein). Positively stained tenocytes were counted, and their distributional differences were verified in 3-dimensional images. RESULTS: The repaired intrasynovial tendons exhibited generally greater apoptosis in the surface region than in the core. The differences were more remarkable in the extended region than in the junction region of the cut tendon. At the core of the junction site, apoptosis of tenocytes was pronounced at all time points, but it was less severe at the core of the extended region. The proliferating cell nuclear antigen-positive and Bcl-2-positive tenocytes decreased significantly and continually at days 28, 42, and 56, respectively; these tenocytes were at a minimum at days 56 and 84. CONCLUSIONS: Apoptotic changes of tenocytes are most marked in the surface region and in the junction region of the healing tendon in the middle and late healing stages. Apoptosis in the core is less dramatic compared to that in the surface in the extended tendon regions. Cellular proliferation declines drastically and is minimal at days 56 and 84. CLINICAL RELEVANCE: Tenocyte apoptosis in the middle and late stages might be an important event contributing to intrasynovial tendon remodeling, which affects the healing strength and formation of adhesions.

Dietary iodine intake in the Chinese population.

OBJECTIVE: To evaluate dietary iodine intake and its potential risks among the Chinese population. METHODS: Individual dietary iodine intake was calculated using food consumption data multiplying by iodine concentration in foods, table salt and drinking water, followed by summing, and then compared with the corresponding age-specific reference values, including Upper Intake Level (UL) and Recommended Nutrient Intake (RNI). RESULTS: In areas with water iodine concentration (WI) lower than 150 µg/L, 80.8% of residents had iodine intake between the RNI and UL, 5.8% higher than UL, and the remaining (13.4%) lower than RNI if iodized salt was consumed. However, in the uniodized salt consumption scenario, only 1.0% of residents between RNI and UL, 1.4% higher than UL, and a large part of residents (97.6%) lower than RNI. In areas with WI higher than 150 µg/L, all residents had iodine intake between RNI and UL if iodized salt was consumed, except 10.5% and 24.9% of residents higher than UL in areas with WI at 150-300 µg/L and higher than 300 µg/L respectively. However, in the uniodized salt consumption scenario, only 1.5% and 1.7% of residents had higher iodine intake than UL respectively. CONCLUSION: The findings suggested that in general, the dietary iodine intake by the Chinese population was appropriate and safe at the present stage. People in areas with WI lower than 150 µg/L were more likely to have iodine deficiency. While people in areas with WI higher than 150 µg/L were more likely to have excessive iodine intake if iodized salt was consumed.

Type I methionine aminopeptidase from Saccharomyces cerevisiae is a potential target for antifungal drug screening.

AIM: To screen antifungal drug candidates using in vitro and in vivo assays based on type I methionine aminopeptidase from Saccharomyces cerevisiae (ScMetAP1). METHODS: A colorimetric assay suitable for high throughput screening (HTS) using recombinant ScMetAP1 protein expressed in Escherichia coli was established for antifungal lead discovery. A series of pyridine-2-carboxylic acid derivatives were characterized and a chemical library of 12,800 pure organic compounds was screened with the in vitro ScMetAP1 assay. Active compounds from the in vitro assay were further evaluated by a growth inhibition assay on yeast strain with deletion of ScMetAP1 gene map1 in comparison with the wild-type yeast strain and the yeast strain with deletion of type II enzyme (ScMetAP2) gene map2. RESULTS: Active ScMetAP1 inhibitors were identified from HTS. Some of the pyridine-2-carboxylic acid derivatives (compound 2 and 3) had selective inhibition of the growth of map2 deletion yeast and weak inhibition on wild-type yeast growth, while no inhibition on map1 deletion yeast. CONCLUSION: ScMetAP1 is a novel potential target for developing antifungal drugs. The in vitro and in vivo ScMetAP1 assays can serve as tools in discovering antifungal drug candidates.

[Introduction of foreign DNA into the genome of rare minnow by sperm electroporation].

Gene transfer was investigated in rare minnow via electroporated sperm. The sperm of the fish mixed with linear DNA (pCAhLFc) was electroporated. Then mature eggs were in vitro fertilized with these sperm cells. The DNA was extracted from the fries and analyzed by PCR. The percentage of fries with foreign genes varied between 25.5% and 66.7%. The observation of the electroporated sperm under microscope indicates that both the vigor of sperms and the successful ratio of the fertilization diminish, indicating that the electroporation with various parameters can hurt the sperm on different extent. The sperm cells electroporated with foreign genes, then was treated by DNA, and the DNA was extracted and was analyzed by PCR. It was found that foreign gene still exists, which approves that the electroporation can force the foreign gene enter the sperm of rare minnow.