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.

Effect of loading speed on gap resistance and tensile strength of flexor tendon repair under cyclic loading test.

Postoperative digit motion is important for the functional recovery of injured tendons. To date, it is unknown whether the loading speed impacts the biomechanical properties of a repaired tendon. This study investigated the effect of loading speed on the gap resistance and tensile strength of tendon repairs. One hundred porcine flexor tendons were repaired with two core sutures, 4-strand modified Kessler and double Q, and cyclically loaded at the speeds of 10, 40, 80, 160, and 320 mm/min. The number of tendons that formed an initial or 2 mm gap at the repair site during cyclic loading, stiffness at the 1st and 20th loading cycles, gap size between tendon ends when cyclic loading ended, and the ultimate strength were recorded. Under the lowest loading speed, the tendons repaired with the 4-strand modified Kessler suture developed significantly larger gaps and smaller stiffness than those with a greater loading speed. The loading speed did not affect the maximum strength of both tendon repairs. The findings suggest that very slow motion promotes gap formation of tendon repair with inferior gap resistance. The rate corresponds to regular hand action or the tendon core suture possessing a strong gap resistance increases the safety margin during early active finger movement. Our findings help to guide the exercise regimens after tendon surgery.

Interference in melanoma CD248 function reduces vascular mimicry and metastasis.

BACKGROUND: Tumor vascular mimicry is an emerging issue that affects patient survival while having no treatment at the current moment. Despite several factors implicated in vascular mimicry, little is known about stromal factors that modulate tumor microenvironment and shape malignant transformation. CD248, a type-I transmembrane protein dominantly expressed in stromal cells, mediates the interaction between cells and extracellular matrix proteins. CD248 protein expression is associated with the metastatic melanoma phenotype and promotes tumor progression in the stromal cells. This study aimed to explore the cell-autonomous effects of CD248 in melanoma vascular mimicry to aid cancer therapy development. METHODS: Loss-of-function approaches in B16F10 melanoma cells were used to study the cell-autonomous effects of CD248 on cell adhesion, migration, proliferation, and vascular mimicry. A solid-phase binding assay was performed to identify the interaction between CD248 and fibronectin. Horizontal and vertical cell migration assays were performed to analyze cell migration activity, and cell-patterned network formation on Matrigel was used to evaluate vascular mimicry activity. Recombinant CD248 (rCD248) proteins were generated, and whether rCD248 interfered with melanoma CD248 functions was evaluated in vitro. An experimental lung metastasis mouse model was used to investigate the effect of rCD248 treatment in vivo. RESULTS: CD248 protein expression in melanoma cells was increased by a fibroblast-conditioned medium. Knockdown of CD248 expression significantly decreased cell adhesion to fibronectin, cell migration, and vascular mimicry in melanoma cells. The lectin domain of CD248 was directly involved in the interaction between CD248 and fibronectin. Furthermore, rCD248 proteins containing its lectin domain inhibited cell adhesion to fibronectin and slowed down cell migration and vascular mimicry. Treatment with rCD248 protein could reduce pulmonary tumor burden, accompanied by a reduction in vascular mimicry in mice with melanoma lung metastasis. CONCLUSION: CD248 expression in melanoma cells promotes malignant transformation by increasing the activity of cell adhesion, migration, and vascular mimicry, whereas rCD248 protein functions as a molecular decoy interfering with tumor-promoting effects of CD248 in melanoma cells.

Effects of a Q Suture Technique as a Core Suture on Resistance to Gap Formation and Tensile Strength in an Ex Vivo Porcine Flexor Tendon Model.

PURPOSE: The Q suture has been reported to be an effective alternative to conventional peripheral sutures in tendon repair. Whether the Q method can be used as a core suture rather than a peripheral suture by adjusting the purchase length is unknown. We tested a double Q suture technique with variable purchase length and studied its effects on gap formation and tensile strength using an ex vivo model. METHODS: Forty pig flexor tendons were repaired using the double Q sutures with purchase lengths of 2, 4, 6, and 8 mm. Twenty tendons repaired using the double Tsuge and double Kessler sutures with an 8-mm purchase length were used as controls. The tendons were subjected to cyclic loading and load-to-failure. The number of tendons that formed an initial or 2-mm gap between the tendon ends, gap distance at the repair site, stiffness, and ultimate strength were recorded. RESULTS: During cyclic loading, the double Q suture with a 4-8-mm purchase had fewer tendons form an initial or 2-mm gap and a smaller gap size at the repair site than the Tsuge and Kessler sutures. The stiffness of the double Q suture with a 6-8-mm purchase length and Tsuge suture was greater than those of the double Kessler suture. The double Q suture with a 2-mm purchase length had smaller ultimate strength than the other sutures. CONCLUSIONS: The Q suture may be an effective tendon repair method whose role can be converted between peripheral and core sutures via adjusting the suture purchase length. With an optimal suture length of 4-6 mm, the double Q method had tensile resistance superior to 4-strand core sutures. CLINICAL RELEVANCE: The double Q suture may be a viable option as a core suture in flexor tendon repair when the purchase length is appropriately adjusted.

Towards intelligent and integrated pest management through an AIoT-based monitoring system.

BACKGROUND: Main bottleneck in facilitating integrated pest management (IPM) is the unavailability of reliable and immediate crop damage data. Without sufficient insect pest and plant disease information, farm managers are unable to make proper decisions to prevent crop damage. This work aims to present how an integrated system was able to drive farm managers towards sustainable and data-driven IPM. RESULTS: A system called Intelligent and Integrated Pest and Disease Management (I2 PDM) system was developed. Edge computing devices were developed to automatically detect and recognize major greenhouse insect pests such as thrips (Frankliniella intonsa, Thrips hawaiiensis, and Thrips tabaci), and whiteflies (Bemisia argentifolii and Trialeurodes vaporariorum), to name a few, and measure environmental conditions including temperature, humidity, and light intensity, and send data to a remote server. The system has been installed in greenhouses producing tomatoes and orchids for gathering long-term spatiotemporal insect pest count and environmental data, for as long as 1368 days. The findings demonstrated that the proposed system supported the farm managers in performing IPM-related tasks. Significant yearly reductions in insect pest count as high as 50.7% were observed on the farms. CONCLUSION: It was concluded that novel and efficient strategies can be achieved by using an intelligent IPM system, opening IPM to potential benefits that cannot be easily realized with a traditional IPM program. This is the first work that reports the development of an intelligent strategic model for IPM based on actual automatically collected long-term data. The work presented herein can help in encouraging farm managers, researchers, experts, and industries to work together in implementing sustainable and data-driven IPM. © 2022 Society of Chemical Industry.

Basic science approaches to common hand surgery problems.

The field of hand surgery is constantly evolving to meet challenges of populations with increasing age and higher demands for active living. While our surgical care has improved over the last decades, it seems that future major improvement in outcomes of clinical treatment will come through advances in biologics and the translation of major discoveries in basic science. This article aims to provide an update on where basic science solutions may answer some of the most critical issues in hand surgery, with a focus on augmentation of tissue repair.

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.

The impact of transverse components on resistance and ultimate strength of 6-strand tendon repairs.

We assessed the effects of tendon core sutures' transverse components on the tensile resistance of two commonly used 6-strand tendon repairs. Tang and Yoshizu #1 repairs (6-strand) were tested and compared with 4-strand rectangular and double Kessler sutures (4-strand). A total of 40 pig flexor tendons were tested under cyclic loading. We recorded the number of tendons that formed a 2-mm gap between two tendon ends during 20 cycles of cyclic loading test, stiffness at the 1st and 20th loading cycle, and gap distance at the repair site and the ultimate repair strength at the 20th cycles. We found that the Yoshizu #1 repairs were more prone to form gaps and their ultimate strength was significantly lower than that of the Tang repair. The transverse components in a 6-strand repair affect gap formation and failure strength.

Time-resolved RNA-seq provided a new understanding of intestinal immune response of European eel (Anguilla anguilla) following infection with Aeromonas hydrophila.

No studies systematically examined the intestinal immune response for yellow stage of European eel (Anguilla anguilla) with Aeromonas hydrophila infection by time-resolved RNA-seq. Here, we examined transcriptional profiles of the intestines at three-time points following infection with A. hydrophila. Intraperitoneal injections caused mortalities within 48 h post-injection (hpi), with the survival rate 87.5% at 24 hpi and 83.9% at 48 hpi. The result from KEGG pathway enrichment analysis showed that the immune related "cytosolic DNA-sensing pathway" was significantly enriched at the first and second time points (6 hpi and 18 hpi), with the up-regulated expression of irf3, il1b, tnfaip3, cxcl8a, ap1-2, c-fos, polr3d, polr3g and polr3k both at 6 hpi and 18 hpi, but not at the third time point (36 hpi). According to the KEGG annotation, 326 immune and inflammation-related DEGs were found. The co-expression network of those 326 DEGs revealed the existence of three modules, and tlr1 was found to be in the center of the biggest module which contained massive DEGs from "signal transduction" and "transport and catabolism". The c3 isoforms showed different expression pattern among the three time points, indicating a unique activation of complement systems at 18 hpi. Furthermore, two cathelicidins (aaCATH_1 and aaCATH_2) were highly up-regulated at the first two time points, and the bacterial growth inhibition assay revealed their antibacterial properties against A. hydrophila. Our data indicated the important roles of cytosolic DNA-sensing pathway, as well as transcripts including tlr1, c3, polr and cathelicidins in the intestine of A. anguilla in response to A. hydrophila infection. The present study will provide leads for functional studies of host-pathogen interactions.

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.

Basic FGF or VEGF gene therapy corrects insufficiency in the intrinsic healing capacity of tendons.

Tendon injury during limb motion is common. Damaged tendons heal poorly and frequently undergo unpredictable ruptures or impaired motion due to insufficient innate healing capacity. By basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) gene therapy via adeno-associated viral type-2 (AAV2) vector to produce supernormal amount of bFGF or VEGF intrinsically in the tendon, we effectively corrected the insufficiency of the tendon healing capacity. This therapeutic approach (1) resulted in substantial amelioration of the low growth factor activity with significant increases in bFGF or VEGF from weeks 4 to 6 in the treated tendons (p < 0.05 or p < 0.01), (2) significantly promoted production of type I collagen and other extracellular molecules (p < 0.01) and accelerated cellular proliferation, and (3) significantly increased tendon strength by 68-91% from week 2 after AAV2-bFGF treatment and by 82-210% from week 3 after AAV2-VEGF compared with that of the controls (p < 0.05 or p < 0.01). Moreover, the transgene expression dissipated after healing was complete. These findings show that the gene transfers provide an optimistic solution to the insufficiencies of the intrinsic healing capacity of the tendon and offers an effective therapeutic possibility for patients with tendon disunion.

Gene therapy strategies to improve strength and quality of flexor tendon healing.

INTRODUCTION: Rupture of the repair and adhesion around a tendon are two major problems after tendon surgery. Novel biological therapies which enhance healing and reduce adhesions are goals of many investigations. Gene therapy offers a new and promising approach to tackle these difficult problems. In the past decade, we sought to develop methods to augment tendon healing and reduce tendon adhesion through gene therapy. AREAS COVERED: This review discusses the methods and results of adeno-associated viral (AAV) type 2 vector gene therapy to increase tendon healing strength and reduce adhesions in a chicken model. Micro-RNA related gene therapy is also discussed. We also developed a controlled release system, which incorporates nanoparticles to deliver micro-RNAs to regulate tendon healing. EXPERT OPINION: We obtained promising results of enhancement of tendon healing strength in a chicken model using AAV2-mediated gene transfer. AAV2-mediated micro-RNA transfer also limited adhesions around the tendon. Controlled release systems incorporating nanoparticles have ideally delivered genes to the healing tendons and resulted in a moderate (but incomplete) reduction of adhesions. It remains to be determined what the best doses are and what other factors are in play in adhesion formation. These are two targets in our future investigations.