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This study aimed to design a novel electrode for reducing tissue thermal damage in radiofrequency-induced intestinal anastomosis. We developed and compared two electrodes (Ring electrode, and Plum electrode with reduced section of the middle fusion area by nearly 80% arising from novel structural design) by performing ex-vivo experiments and finite element analysis. In contrast to the Ring electrode group, slightly higher mean strength is acquired with the tensile force and burst pressure results increasing from 9.7 ± 1.47 N, 84.0 ± 5.99 mmHg to 11.1 ± 1.71 N, 89.4 ± 6.60 mmHg, respectively, as well as a significant reduction in tissue thermal damage for the Plum electrode group, with compression pressure of 20 kPa, RF energy of 120 W and welding duration of 8 s applied to the target regions to achieve anastomosis. Besides, the novel structural design of the Plum electrode can counteract the tension generated by intestinal peristalsis and enhance the biomechanical strength of the anastomotic area. The histological observation showed that the fusion area of the two-layer intestinal tissue is tightly connected with decreased thickness. The novel electrode (Plum electrode) could reduce tissue thermal damage in radiofrequency-induced intestinal anastomosis. [ABSTRACT FROM AUTHOR]

For decades, radiofrequency (RF)-induced tissue fusion has garnered great attention due to its potential to replace sutures and staples for anastomosis of tissue reconstruction. However, the complexities of achieving high bonding strength and reducing excessive thermal damage present substantial limitations of existing fusion devices. This study proposed a discrete linkage-type electrode to carry out ex vivo RF-induced intestinal anastomosis experiments. The anastomotic strength was examined by burst pressure and shear strength test. The degree of thermal damage was monitored through an infrared thermal imager. And the anastomotic stoma fused by the electrode was further investigated through histopathological and ultrastructural observation. The burst pressure and shear strength of anastomotic tissue can reach 62.2 ± 3.08 mmHg and 8.73 ± 1.11N, respectively, when the pressure, power and duration are 995 kPa, 160 W and 13 s, and the thermal damage can be controlled within limits. Histopathological and ultrastructural observation indicate that an intact and fully fused stomas with collagenic crosslink can be formed. The discrete linkage-type electrode presents favorable efficiency and security in RF-induced tissue fusion, and these results are informative to the design of electrosurgical medical devices with controllable pressure and energy delivery. [ABSTRACT FROM AUTHOR]

This study aimed to develop a novel electrode to achieve balance between anastomotic strength and tissue thermal damage for radiofrequency-induced intestinal anastomosis. Methods: The mechanical properties of the novel electrode were analyzed by finite element method, and then the temperature and thermal damage distribution of intestinal tissue during welding process were analyzed by electric-thermalmechanical multi-field coupled finite element analysis. In ex vivo experiments, the biomechanical strength of anastomotic area was assessed by indexes of tensile force and burst pressure. A thermocouple probe and an infrared thermal imager were used to monitor the temperature and thermal damage of the intestinal tissue in experiments. Furthermore, histopathological examination and transmission electron microscopy observation were used to observe the morphology and microstructure of anastomotic area. Results: A slightly higher mean biomechanical strength is acquired with the tensile force and burst pressure results increasing from 9.7 ± 1.47 N, 84.0 ± 5.99 mmHg to 11.8 ± 2.01 N, 89.6 ± 6.79 mmHg, respectively, as well as the percentage of necrotic tissue caused by thermal damage decreasing from 89% to 33% for the novel electrode group, with compression force of 20 N, radiofrequency (RF) energy of 120 W and welding duration of 8 s applied to the target regions to achieve anastomosis. Moreover, tightly connected intestinal tissue with collagenic crosslink in the fusion area could be observed by histopathological examination and transmission electron microscopy. Conclusions: Our study shows that the proposed novel electrode could achieve balance between anastomotic strength and tissue thermal damage for radiofrequencyinduced intestinal anastomosis. [ABSTRACT FROM AUTHOR]

Background: Tissue welding is an electrosurgical technique that can fuse tissue for small intestine anastomosis. However, limited knowledge exists on its application in mucosa-mucosa end-to-end anastomosis. This study investigates the effects of initial compression pressure, out-put power, and duration time on anastomosis strength ex vivo in mucosa-mucosa end-to-end anastomosis.Methods:Ex vivo porcine bowel segments were used to create 140 mucosa-mucosa end-to-end fusions. Different experimental parameters were employed for fusion, including initial com-pression pressure (50kPa–400 kPa), output power (90W, 110W, and 140W), and fusion time (5, 10, 15, 20 s). The fusion quality was measured by burst pressure and optical microscopes.Results: The best fusion quality was achieved with an initial compressive pressure between 200 and 250 kPa, an output power of 140W, and a fusion time of 15 s. However, an increase in output power and duration time resulted in a wider range of thermal damage. There was no significant difference between the burst pressure at 15 and 20 s (p > 0.05). However, a substantial increase in thermal damage was observed with longer fusion times of 15 and 20 s (p < 0.05).Conclusion: The best fusion quality for mucosa-mucosa end-to-end anastomosis ex vivo is achieved when the initial compressive pressure is between 200 and 250 kPa, the output power is approximately 140W, and the fusion time is approximately 15 s. These findings can serve as a valuable theoretical foundation and technical guidance for conducting animal experiments in vivo and subsequent tissue regeneration.

Radiofrequency (RF)-induced tissue fusion shows great potential in sealing intestinal tissue without foreign materials. To improve the performance of RF-induced tissue fusion, a novel self-cooling jaw has been designed to minimize thermal damage during the fusion. The prototype of self-cooling jaws was developed and manufactured. A total number of 60 mucosa-to-mucosa fusions were conducted using ex-vivo porcine intestinal segments with the proposed design and conventional bipolar jaws. The effects of intestinal fusion were evaluated based on temperature curves, burst pressure, thermal damage, and histological appearances. The self-cooling jaws showed significant decrease in temperature during the fusion process. An optimal burst pressure (5.7 ± 0.5 kPa) and thermal damage range (0.9 ± 0.1 mm) were observed when the applied RF power was 100 W. The thermal damage range of the prototype has almost decreased 36% in comparison with the conventional bipolar jaws (1.4 ± 0.1 mm). The histological observation revealed that a decrease of thermal damage was achieved through the application of self-cooling jaws. The self-cooling jaws were proved to be effective for reducing the thermal damage during RF-induced tissue fusion, which could potentially promote the clinical application of tissue fusion techniques in the future. [ABSTRACT FROM AUTHOR]

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Epithelial remodelling plays a crucial role during development. The ability of epithelial sheets to temporarily lose their integrity as they fuse with other epithelial sheets underpins events such as the closure of the neural tube and palate. During fusion, epithelial cells undergo some degree of epithelial-mesenchymal transition (EMT), whereby cells from opposing sheets dissolve existing cell-cell junctions, degrade the basement membrane, extend motile processes to contact each other, and then re-establish cell-cell junctions as they fuse. Similar events occur when an epithelium is wounded. Cells at the edge of the wound undergo a partial EMT and migrate towards each other to close the gap. In this review, we highlight the emerging role of Netrins in these processes, and provide insights into the possible signalling pathways involved. Netrins are secreted, laminin-like proteins that are evolutionarily conserved throughout the animal kingdom. Although best known as axonal chemotropic guidance molecules, Netrins also regulate epithelial cells. For example, Netrins regulate branching morphogenesis of the lung and mammary gland, and promote EMT during Drosophila wing eversion. Netrins also control epithelial fusion during optic fissure closure and inner ear formation, and are strongly implicated in neural tube closure and secondary palate closure. Netrins are also upregulated in response to organ damage and epithelial wounding, and can protect against ischemia-reperfusion injury and speed wound healing in cornea and skin. Since Netrins also have immunomodulatory properties, and can promote angiogenesis and re-innervation, they hold great promise as potential factors in future wound healing therapies. [ABSTRACT FROM AUTHOR]

While decades of research have enriched the knowledge of how to grow cells into mature tissues, little is yet known about the next phase: fusing of these engineered tissues into larger functional structures. The specific effect of multicellular interfaces on tissue fusion remains largely unexplored. Here, a facile 3D‐bioassembly platform is introduced to primarily study fusion of cartilage–cartilage interfaces using spheroids formed from human mesenchymal stromal cells (hMSCs) and articular chondrocytes (hACs). 3D‐bioassembly of two adjacent hMSCs spheroids displays coordinated migration and noteworthy matrix deposition while the interface between two hAC tissues lacks both cells and type‐II collagen. Cocultures contribute to increased phenotypic stability in the fusion region while close initial contact between hMSCs and hACs (mixed) yields superior hyaline differentiation over more distant, indirect cocultures. This reduced ability of potent hMSCs to fuse with mature hAC tissue further underlines the major clinical challenge that is integration. Together, this data offer the first proof of an in vitro 3D‐model to reliably study lateral fusion mechanisms between multicellular spheroids and mature cartilage tissues. Ultimately, this high‐throughput 3D‐bioassembly model provides a bridge between understanding cellular differentiation and tissue fusion and offers the potential to probe fundamental biological mechanisms that underpin organogenesis. [ABSTRACT FROM AUTHOR]

Defects in choroid fissure (CF) formation and closure lead to coloboma, a major cause of childhood blindness. Despite genetic advances, the cellular defects underlying coloboma remain poorly elucidated due to our limited understanding of normal CF morphogenesis. We address this deficit by conducting high-resolution spatio-temporal analyses of CF formation and closure in the chick, mouse and fish. We show that a small ventral midline invagination initiates CF formation in the medial-proximal optic cup, subsequently extending it dorsally toward the lens, and proximally into the optic stalk. Unlike previously supposed, the optic disc does not form solely as a result of this invagination. Morphogenetic events that alter the shape of the proximal optic cup also direct clusters of outer layer and optic stalk cells to form dorsal optic disc. A cross-species comparison suggests that CF closure can be accomplished by breaking down basement membranes (BM) along the CF margins, and by establishing BM continuity along the dorsal and ventral surfaces of the CF. CF closure is subsequently accomplished via two distinct mechanisms: tissue fusion or the intercalation of various tissues into the inter-CF space. We identify several novel cell behaviors that underlie CF fusion, many of which involve remodeling of the retinal epithelium. In addition to BM disruption, these include NCAD downregulation along the SOX2 + retinal CF margin, and the protrusion or movement of partially polarized retinal cells into the inter-CF space to mediate fusion. Proximally, the inter-CF space does not fuse or narrow and is instead loosely packed with migrating SOX2 + /PAX2 + /Vimentin + astrocytes until it is closed by the outgoing optic nerve. Taken together, our results highlight distinct proximal-distal differences in CF morphogenesis and closure and establish detailed cellular models that can be utilized for understanding the genetic bases of coloboma. [ABSTRACT FROM AUTHOR]

Tissue closure involves the coordinated unidirectional movement of a group of cells without loss of cell–cell contact. However, the molecular mechanisms controlling the tissue closure are not fully understood. Here, we demonstrate that Lamin C, the sole A-type lamin in Drosophila , contributes to the process of thorax closure in pupa. High expression of Lamin C was observed at the leading front of the migrating wing imaginal discs. Live imaging analysis revealed that knockdown of Lamin C in the thorax region affected the coordinated movement of the leading front, resulting in incomplete tissue fusion required for formation of the adult thorax. The closure defect due to knockdown of Lamin C correlated with insufficient accumulation of F-actin at the front. Our study indicates a link between A-type lamin and the cell migration behavior during tissue closure. [ABSTRACT FROM AUTHOR]

Ocular coloboma (OC) is a defect in optic fissure closure and is a common cause of severe congenital visual impairment. Bilateral OC is primarily genetically determined and shows marked locus heterogeneity. Whole-exome sequencing (WES) was used to analyze 12 trios (child affected with OC and both unaffected parents). This identified de novo mutations in 10 different genes in eight probands. Three of these genes encoded proteins associated with actin cytoskeleton dynamics: ACTG1, TWF1, and LCP1. Proband-only WES identified a second unrelated individual with isolated OC carrying the same ACTG1 allele, encoding p.(Pro70Leu). Both individuals have normal neurodevelopment with no extra-ocular signs of Baraitser-Winter syndrome. We found this mutant protein to be incapable of incorporation into F-actin. The LCP1 and TWF1 variants each resulted in only minor disturbance of actin interactions, and no further plausibly causative variants were identified in these genes on resequencing 380 unrelated individuals with OC. [ABSTRACT FROM AUTHOR]

BACKGROUND: Recent advances in tissue engineering have led to the development of the concept of bioprinting as an interesting alternative to traditional tissue engineering approaches. Biopaper, a biomimetic hydrogel, is an essential component of the bioprinting process. OBJECTIVE: The aim of this work was to synthesize a biopaper made of fibrin-gelatin hybrid hydrogel for application in skin bioprinting. METHODS: Different composition percentages of the two biopolymer hydrogels, fibrin-gelatin, have been studied for the construction of the biopaper and were examined in terms of water absorption, biodegradability, glucose absorption, mechanical properties and water vapor transmission. Subsequently, tissue fusion study was performed on prepared 3T3 fibroblast cell line pellets embedded into the hydrogel. RESULTS: Based on the obtained results, fibrin-gelatin blend hydrogel with the same proportion of two components provides a natural scaffold for fibroblast-based bioink embedding and culture. CONCLUSIONS: The suggested optimized hydrogel was a suitable candidate as a biopaper for skin bioprinting technology. [ABSTRACT FROM AUTHOR]

Current efforts to evaluate the performance of laparoscopic arterial fusion devices are limited to costly, time consuming, empirical studies. Thus, a finite element (FE) model, with the ability to predict device performance would improve device design and reduce development time and costs. This study introduces a model of the heat transfer through an artery during electrosurgical procedures that accounts for changes in thermal material properties due to water loss and temperature. Experiments then were conducted by applying a known heat and pressure to carefully sectioned pieces of porcine splenic arteries and measuring cut completeness. From this data, equations were developed to predict at which temperature and pressure arterial tissue is cut. These results were then incorporated into a fully coupled thermomechanical FE model with the ability to predict whole artery cutting. An additional experiment, performed to examine the accuracy of the model, showed that the model predicted complete artery cut results correctly in 28 of 32 tests. The predictive ability of this FE model opens a gateway to more advanced electrosurgical fusion devices and modeling techniques of electrosurgical procedures by allowing for faster, cheaper and more comprehensive device design. [ABSTRACT FROM AUTHOR]

Purpose:Bipolar radio-frequency-induced thermofusion (BiRTh) of intestinal tissue might replace conventional stapling devices which are associated with technical and functional complications. Previous results of our study group confirmed the feasibility to fuse intestinal tissue using BiRTh-induced thermofusionex vivo.The aim of this study was now to evaluate the efficacy of fusing intestinal tissuein vivoby BiRTh-induced thermofusion. Materials and methods:In male Wistar rats a blind bowel originating from the caecum was closed either by BiRTh (n = 24) or conventional suture (n = 16). At 6 h, 48 h, 4 days, and 2 weeks after the procedure caecum bursting pressure was measured to compare both groups. Results:In total 18 of 21 (85.7%) thermofused and 15 of 16 (93.7%) sutured cecal stumps were primarily tight and leakage-proof (p >0.05). The operative time was comparable in both groups without significant differences. Both groups showed increases in bursting pressure over the post-operative period. The mean bursting pressure for thermofusion was 47.8, 48.3, 55.2, and 68.0 mmHg, compared to 69.8, 51.5, 70.0 and 71.0 mmHg in the hand-sutured group (p > 0.05) after 6 h, 48 h, 4 days, and 2 weeks, respectively. Conclusion:These results suggest that BiRTh-induced thermofusion is a safe and feasible method for fusing intestinal tissue in this experimentalin vivomodel and could be an innovative approach for achieving gastrointestinal anastomoses. [ABSTRACT FROM PUBLISHER]

There are three major groups of parasitic platyhelminths (flatworms). The digeneans and cestodes are endoparasites, while the monogeneans are ectoparasites mostly on the gills or skin of fishes. Monogeneans are hermaphrodite and, with the exception of the gyrodactylids, mostly protandrous, the male reproductive system maturing before the female system. Their ectoparasitic life-style provides unique opportunities to observe the reproductive biology of living platyhelminths, opportunities restricted in digeneans and cestodes by their endoparasitic habits. Moreover, the male copulatory organs (MCOs) of monogeneans are of special interest because of their perplexing diversity, ranging from sclerotised penis tubes, many with accessory sclerites, to cirruses and genital atrium armature (hooks and spines). The relatively few accounts in the literature of mating in monogeneans are reproduced in this review, together with consideration of the following aspects of sperm transfer: structure and function of MCOs; self-insemination; spermatophores and pseudospermatophores; "hypodermic" and transtegumental insemination; tissue fusion; glands associated with MCOs and vaginae; finding a mating partn [ABSTRACT FROM AUTHOR]

Tissue fusion eliminates physical voids in a tissue to forma continuous structure and is central to many processes in development and repair. Fusion events in vivo, particularly in embryonic development, often involve the purse-string contraction of a pluricellular actomyosin cable at the free edge. However, in vitro, adhesion of the cells to their substrate favors a closure mechanism mediated by lamellipodial protrusions, which has prevented a systematic study of the purse-string mechanism. Here, we show that monolayers can cover well-controlled mesoscopic nonadherent areas much larger than a cell size by purse-string closure and that active epithelial fluctuations are required for this process. We have formulated a simple stochastic model that includes purse-string contractility, tissue fluctuations, and effective friction to qualitatively and quantitatively account for the dynamics of closure. Our data suggest that, in vivo, tissue fusion adapts to the local environment by coordinating lamellipodial protrusions and purse-string contractions. [ABSTRACT FROM AUTHOR]

Background: Advanced bipolar radiofrequency (RF) energy-based devices are increasingly used for non-vascular tissue sealing, transection and anastomosis. Although the potential to further develop this technology is clear, the limitations of commercially available devices are poorly understood. This systematic review examines the current utilisation of advanced bipolar RF fusion technology for non-vascular applications in thoracic and abdominal tissues and organs. Methods: Medline, Embase, Web of Science and Cochrane library databases were searched. Studies examining the non-vascular application of advanced bipolar RF technology in the abdomen and thorax were assessed. Measurement of seal sufficiency and the extent of tissue injury induced by bipolar RF energy application were the main outcomes of interest. Results: Forty-six animal and human studies conducted in and ex vivo across a range of tissue types, met the inclusion criteria. The diversity of study protocols together with the heterogeneity of their outcomes prevented pooled analysis. However, the experimental animal studies assessed suggest that bipolar RF fusion devices are capable of effectively sealing most tissue types with the notable exception of large caliber bronchi (>4 mm) and bile ducts, where the rate of seal failure was unacceptably high. Human studies were additionally found to predominantly use bipolar RF technology as a means of parenchymal and ductal occlusion. A similar trend was seen amongst animal studies with only a handful of papers examining bipolar RF energy use for (bowel) anastomosis. Conclusion: Bipolar RF fusion devices are capable of safely sealing a variety of thoracic and anterior abdominal tissues with an injury and leakage profile comparable to established technologies. Although thermal monitoring is increasingly sophisticated and multimodal, consistent real time tracking of a multitude of parameters is necessary in order to expand RF fusion technology utilisation to complete an array of tasks such as vessel ligation, ductal obliteration and anastomosis in the open and laparoscopic environments. [ABSTRACT FROM AUTHOR]

Zippering is a phenomenon of tissue morphogenesis whereby fusion between opposing epithelia progresses unidirectionally over significant distances, similar to the travel of a zip fastener, to ultimately ensure closure of an opening. A comparable process can be observed during Drosophila dorsal closure and mammalian wound healing, while zippering is employed by numerous organs such as the optic fissure, palatal shelves, tracheoesophageal foregut, and presumptive genitalia to mediate tissue sealing during normal embryonic development. Particularly striking is zippering propagation during neural tube morphogenesis, where the fusion point travels extensively along the embryonic axis to ensure closure of the neural tube. Advances in time-lapse microscopy and culture conditions have opened the opportunity for successful imaging of whole-mouse embryo development over time, providing insights into the precise cellular behavior underlying zippering propagation. Studies in mouse and the ascidian Ciona have revealed the fine-tuned cell shape changes and junction remodeling which occur at the site of zippering during neural tube morphogenesis. Here, we describe a step-by-step method for imaging at single-cell resolution the process of zippering and tissue remodeling which occurs during closure of the spinal neural tube in mouse. We also provide instructions and suggestions for quantitative morphometric analysis of cell behavior during zippering progression. This procedure can be further combined with genetic mutant models (e.g., knockouts), offering the possibility of studying the dynamics of tissue fusion and zippering propagation, which underlie a wide range of open neural tube defects., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cellular spheroids were investigated as tissue-engineered building blocks that can be fused to form functional tissue constructs. While spheroids can be assembled using passive contacts for the fusion of complex tissues, physical forces can be used to promote active contacts to improve tissue homogeneity and accelerate tissue fusion. Understanding the mechanisms affecting the fusion of spheroids is critical to fabricating tissues. Here, manipulation of the spheroid composition was used to accelerate the fusion process mediated by magnetic forces. The Janus structure of magnetic cellular spheroids spatially controls iron oxide magnetic nanoparticles (MNPs) to form two distinct domains: cells and extracellular MNPs. Studies were performed to evaluate the influence of extracellular matrix (ECM) content and cell number on the fusion of Janus magnetic cellular spheroids (JMCSs). Results showed that the integration of iron oxide MNPs into spheroids increased the production of collagen over time when compared to spheroids without MNPs. The results also showed that ring tissues composed of JMCSs with high ECM concentrations and high cell numbers fused together, but exhibited less contraction when compared to their lower concentration counterparts. Results from spheroid fusion in capillary tubes showed that low ECM concentrations and high cell numbers experienced more fusion and cellular intermixing over time when compared to their higher counterparts. These findings indicate that cell–cell and cell–matrix interactions play an important role in regulating fusion, and this understanding sets the rationale of spheroid composition to fabricate larger and more complex tissue-engineered constructs. [ABSTRACT FROM AUTHOR]

Cellular spheroids were studied to determine their use as “bioinks” in the biofabrication of tissue engineered constructs. Specifically, magnetic forces were used to mediate the cyclic longitudinal stretching of tissues composed of Janus magnetic cellular spheroids (JMCSs), as part of a post-processing method for enhancing the deposition and mechanical properties of an extracellular matrix (ECM). The purpose was to accelerate the conventional tissue maturation process via novel post-processing techniques that accelerate the functional, structural, and mechanical mimicking of native tissues. The results of a forty-day study of JMCSs indicated an expression of collagen I, collagen IV, elastin, and fibronectin, which are important vascular ECM proteins. Most notably, the subsequent exposure of fused tissue sheets composed of JMCSs to magnetic forces did not hinder the production of these key proteins. Quantitative results demonstrate that cyclic longitudinal stretching of the tissue sheets mediated by these magnetic forces increased the Young’s modulus and induced collagen fiber alignment over a seven day period, when compared to statically conditioned controls. Specifically, the elastin and collagen content of these dynamically-conditioned sheets were 35- and three-fold greater, respectively, at seven days compared to the statically-conditioned controls at three days. These findings indicate the potential of using magnetic forces in tissue maturation, specifically through the cyclic longitudinal stretching of tissues.

Heat-induced tissue fusion via radio-frequency (RF) energy has gained wide acceptance clinically and here we present the first optical-Raman-spectroscopy study on tissue fusion samples in vitro. This study provides direct insights into tissue constituent and structural changes on the molecular level, exposing spectroscopic evidence for the loss of distinct collagen fibre rich tissue layers as well as the denaturing and restructuring of collagen crosslinks post RF fusion. These findings open the door for more advanced optical feedback-control methods and characterization during heat-induced tissue fusion, which will lead to new clinical applications of this promising technology. [ABSTRACT FROM AUTHOR]

Tissue fusion is a growing area of medical research that enables mechanical closure of tissues without the need of foreign bodies such as sutures or staples. Utilizing heat and pressure applied for a specified time, a bond can be formed between adjacent tissues. The success or failure of tissue fusion is contingent upon the strength of the bond it creates between opposing tissues, yet little characterization has been done to measure the strength of this interface as a function of the input parameters, such as heat and pressure. Previous studies have examined the strength of tissue fusion using clinically relevant outcomes such as bursting pressure or tearing strength, but none have explored metrics more appropriate for determining the mechanics of the actual bond such as peel or shear strengths. The goal of this study is to establish methodology for T-peel and lap shear testing of fused tissues and measure the fusion bonding strength as a function of temperature and time using the ConMed Altrus® laparoscopic thermal fusion device. Across five temperatures (120, 140, 150, 160, 170°C) and four time durations (500, 1000, 1800, 3000ms) the mean peeling strength, ultimate shear strength, and bursting pressure of fused porcine splenic arteries were measured. The shear strength increased with increasing temperature and time with an ultimate shear strength at 160°C and 3000ms equal to 290 ± 99Pa. No trend was observed between the input parameters of time and applied temperature and the mean peeling force, although there were significant differences between groups. The bursting pressure increased significantly with increasing durations, but no trend was noted between temperature and bursting pressure. The shear strength data suggest there is some physical or chemical reaction which occurs in the tissue between 120°C and 150°C which provides a stronger bond. The shear and peel results also reveal that the fusion bond undergoes brittle failure. This study suggests that the tissue fusion bond is maximized at temperatures over 150°C and at a time of 3000ms using the ConMed Altrus® and that input parameters can be tuned to optimize the strength of the bonded region. [ABSTRACT FROM AUTHOR]

Abstract: Cell aggregates, or spheroids, have been used as building blocks to fabricate scaffold-free tissues that can closely mimic the native three-dimensional in vivo environment for broad applications including regenerative medicine and high throughput testing of drugs. The incorporation of magnetic nanoparticles (MNPs) into spheroids permits the manipulation of spheroids into desired shapes, patterns, and tissues using magnetic forces. Current strategies incorporating MNPs often involve cellular uptake, and should therefore be avoided because it induces adverse effects on cell activity, viability, and phenotype. Here, we report a Janus structure of magnetic cellular spheroids (JMCS) with spatial control of MNPs to form two distinct domains: cells and extracellular MNPs. This separation of cells and MNPs within magnetic cellular spheroids was successfully incorporated into cellular spheroids with various cellular and extracellular compositions and contents. The amount of cells that internalized MNPs was quantified and showed that JMCSs resulted in significantly lower internalization (35%) compared to uptake spheroids (83%, p < 0.05). Furthermore, the addition of MNPs to cellular spheroids using the Janus method has no adverse effects on cellular viability up to seven weeks, with spheroids maintaining at least 82% viability over 7 weeks when compared to control spheroids without MNPs. By safely incorporating MNPs into cellular spheroids, results demonstrated that JMCSs were capable of magnetic manipulation, and that magnetic forces used during magnetic force assembly mediate fusion into controlled patterns and complex tissues. Finally, JMCSs were assembled and fused into a vascular tissue construct 5 mm in diameter using magnetic force assembly. [Copyright &y& Elsevier]

Effective cell invasion into thick electrospun biomimetic scaffolds is an unsolved problem. One possible strategy to biofabricate tissue constructs of desirable thickness and material properties without the need for cell invasion is to use thin (<2 µm) porous electrospun meshes and self-assembling (capable of tissue fusion) tissue spheroids as building blocks. Pre-stretched electrospun meshes remained taut in cell culture and were able to support tissue spheroids with minimal deformation. We hypothesize that elastic electrospun scaffolds could be used as temporal support templates for rapid self-assembly of cell spheroids into higher order tissue structures, such as engineered vascular tissue. The aim of this study was to investigate how the attachment of tissue spheroids to pre-stretched polyurethane scaffolds may interfere with the tissue fusion process. Tissue spheroids attached, spread, and fused after being placed on pre-stretched polyurethane electrospun matrices and formed tissue constructs. Efforts to eliminate hole defects with fibrogenic tissue growth factor-β resulted in the increased synthesis of collagen and periostin and a dramatic reduction in hole size and number. In control experiments, tissue spheroids fuse on a non-adhesive hydrogel and form continuous tissue constructs without holes. Our data demonstrate that tissue spheroids attached to thin stretched elastic electrospun scaffolds have an interrupted tissue fusion process. The resulting tissue-engineered construct phenotype is a direct outcome of the delicate balance of the competing physical forces operating during the tissue fusion process at the interface of the pre-stretched elastic scaffold and the attached tissue spheroids. We have shown that with appropriate treatments, this process can be modulated, and thus, a thin pre-stretched elastic polyurethane electrospun scaffold could serve as a supporting template for rapid biofabrication of thick tissue-engineered constructs without the need for cell invasion. [ABSTRACT FROM AUTHOR]

Background: Bipolar radiofrequency (RF) induced tissue fusion is believed to have the potential to seal and anastomose intestinal tissue thereby providing an alternative to current techniques which are associated with technical and functional complications. This study examines the mechanical and cellular effects of RF energy and varying compressive pressures when applied to create ex vivo intestinal seals. Methods: A total of 299 mucosa-to-mucosa fusions were formed on ex vivo porcine small bowel segments using a prototype bipolar RF device powered by a closed-loop, feedback-controlled RF generator. Compressive pressures were increased at 0.05 MPa intervals from 0.00 to 0.49 MPa and RF energy was applied for a set time period to achieve bowel tissue fusion. Seal strength was subsequently assessed using burst pressure and tensile strength testing, whilst morphological changes were determined through light microscopy. To further identify the subcellular tissue changes that occur as a result of RF energy application, the collagen matrix in the fused area of a single bowel segment sealed at an optimal pressure was examined using transmission electron microscopy (TEM). Results: An optimal applied compressive pressure range was observed between 0.10 and 0.25 MPa. Light microscopy demonstrated a step change between fused and unfused tissues but was ineffective in distinguishing between pressure levels once tissues were sealed. Non uniform collagen damage was observed in the sealed tissue area using TEM, with some areas showing complete collagen denaturation and others showing none, despite the seal being complete. This finding has not been described previously in RF-fused tissue and may have implications for in vivo healing. Conclusions: This study shows that both bipolar RF energy and optimal compressive pressures are needed to create strong intestinal seals. This finding suggests that RF fusion technology can be effectively applied for bowel sealing and may lead to the development of novel anastomosis tools. [ABSTRACT FROM AUTHOR]

The development of the embryonic brain critically depends on successfully completing cranial neural tube closure (NTC). Failure to properly close the neural tube results in significant and potentially lethal neural tube defects (NTDs). We believe these malformations are caused by disruptions in normal developmental programs such as those involved in neural plate morphogenesis and patterning, tissue fusion, and coordinated cell behaviors. Cranial NTDs include anencephaly and craniorachischisis, both lethal human birth defects. Newly emerging methods for molecular and cellular analysis offer a deeper understanding of not only the developmental NTC program itself but also mechanical and kinetic aspects of closure that may contribute to cranial NTDs. Clarifying the underlying mechanisms involved in NTC and how they relate to the onset of specific NTDs in various experimental models may help us develop novel intervention strategies to prevent NTDs. [ABSTRACT FROM AUTHOR]

Tissue fusion events during embryonic development are crucial for the correct formation and function of many organs and tissues, including the heart, neural tube, eyes, face and body wall. During tissue fusion, two opposing tissue components approach one another and integrate to form a continuous tissue; disruption of this process leads to a variety of human birth defects. Genetic studies, together with recent advances in the ability to culture developing tissues, have greatly enriched our knowledge of the mechanisms involved in tissue fusion. This review aims to bring together what is currently known about tissue fusion in several developing mammalian organs and highlights some of the questions that remain to be addressed. INSETS: Box. 1. Palatal fusion defects;Box. 2. Neural tube defects;Box. 3. Fusion-related cardiac defects. [ABSTRACT FROM AUTHOR]

Purpose: In recent years, vessel sealing has become a well-established method in surgical practice for sealing and transecting vessels. Since this technology depends on the fusion of collagen fibers abundantly present in the intestinal wall, it should also be possible to create intestinal anastomoses by thermofusion. Bipolar radiofrequency-induced thermofusion of intestinal tissue may replace traditionally used staples or sutures in the future. The aim of this study was to evaluate the feasibility of fusing intestinal tissue ex vivo by bipolar radiofrequency-induced thermofusion. Materials and methods: An experimental setup for temperature-controlled bipolar radiofrequency-induced thermofusion of porcine ( n = 30) and rat ( n = 18) intestinal tissue was developed. Colon samples were harvested and then anastomosed, altering compressive pressure to examine its influence on anastomotic bursting pressure during radiofrequency-induced anastomotic fusion. For comparison, mechanical stapler anastomoses of porcine colonic samples and conventional suturing of rat colonic samples identical to those used for fusion experiments were prepared, and burst pressure was measured. Results: All thermofused colonic anastomoses were primarily tight and leakage proof. For porcine colonic samples, an optimal interval of compressive pressure (1,125 mN/mm) with respect to a high amount of burst pressure (41 mmHg) was detected. The mean bursting pressure for mechanical stapler anastomosis was 60.7 mmHg and did not differ from the thermofusion ( p = 0.15). Furthermore, the mean bursting pressure for thermofusion of rat colonic samples was up to 69.5 mmHg for a compressive pressure of 140 mN/mm. Conclusion: These results confirm the feasibility to create experimental intestinal anastomoses using bipolar radiofrequency-induced thermofusion. The stability of the induced thermofusion showed no differences when compared to that of conventional anastomoses. Bipolar radiofrequency-induced thermofusion of intestinal tissue represents an innovative approach for achieving gastrointestinal anastomoses. [ABSTRACT FROM AUTHOR]

Background: Radiofrequency-controlled tissue fusion is a novel technology but the associated lateral thermal damage has not been determined. Methods: Lateral thermal spread of in vivo and ex vivo bowel in a live porcine model fused by radiofrequency energy was evaluated using dynamic infrared thermography and histology. Results: Mean maximum thermal spread measured on histology was <1.2 mm, with no significant difference between thermal spreads for in vivo and ex vivo bowel for radiofrequency energy delivered at 50 V ( p = 0.98) and 100 V ( p = 0.85). Mean total maximum thermal spread measured by dynamic infrared thermography was <3.9 mm wide on both sides of the instrument with no significant difference between thermal spreads for in vivo and ex vivo bowel for radiofrequency energy delivered at 50 V ( p = 0.34) and 100 V ( p = 0.19). Fusion quality for in vivo tissue was better when radiofrequency energy was delivered at 100 V compared with 50 V. However, thermal spread measurements and maximum temperatures reached in the tissue were similar in well- and poorly fused bowel. Thermal changes in well-fused bowel were more uniform throughout the different bowel wall layers, whereas in poorly fused tissues, the mucosa did not show thermally induced changes. There were no significant differences between the maximum temperatures detected for in vivo and ex vivo bowel for radiofrequency energy delivered at 50 V ( p = 0.25) and 100 V ( p = 0.14). Conclusions: The total thermal changes at both sides of fused bowel are <3.9 mm. The heat sink effect of the application instrument overshadowed any effects of perfusion on limiting thermal spread. Also, using greater amounts of radiofrequency energy at 100 V to achieve better quality fusion does not necessarily increase lateral thermal damage compared with 50 V. [ABSTRACT FROM AUTHOR]

Tubular tissue constructs prepared from neonatal human dermal fibroblasts entrapped in fibrin gel were incubated on a mandrel for three weeks to allow for initial fibrin remodeling into tissue before being concentrically layered and incubated for an additional three weeks on the mandrel. Upon harvest, double layer constructs were not statistically different from single layer control constructs in terms of length, collagen density, cell density, tensile modulus, or ultimate tensile strength. However, the thickness and burst pressure were both approximately twice the single layer control values. Metabolically active cells were detected at the interface, and scanning electron microscopy revealed fiber structures bridging the two layers, co-localizing with the cells, which exhibited minimal migration across the layers. In contrast, double layer constructs where tissue fusion was prohibited by mechanical distraction of the layers showed no increase in burst pressure despite having increased thickness and the same collagen and cell densities of the single layer control constructs; moreover, the burst failure occurred sequentially in the layers in contrast to simultaneous failure for the fused double layer constructs. This study provides insight into the nature of the interface and the role of cell behavior when tissue fusion occurs between two layers of bioartificial tissue in vitro. It also suggests a method for improving the burst strength of fibrin-based tubular tissue constructs by increasing the construct thickness via concentrically layering and fusing two constructs. [ABSTRACT FROM AUTHOR]

Vessel sealing has been well-established in surgical practice in recent years. Bipolar radiofrequency-induced thermofusion (BIRTH) of intestinal tissue might replace traditionally used staples or sutures in the near future. In this experimental study, the influence of compressive pressure, fusion temperature, and duration of heating on the quality of intestinal anastomosis was investigated to obtain the relevant major parameters for the in vivo use of this system. An experimental setup for a closed-loop temperature-controlled bipolar radiofrequency-induced thermofusion of porcine intestinal tissue was developed. Twenty-four colon samples were harvested from nine different Saalower-Kräuter pigs and then anastomosed altering compressive pressure on five different levels to explore its influence on anastomotic bursting pressure. The anastomotic bursting strength depends on the compressive pressure applied to the colonic fusion site. An optimal interval of compressive pressure (CP = 1.125 N/mm2) in respect of a high amount of burst pressure was detected. A correlation ( r = 0.54, p = 0.015) of burst pressure to delta compression indicated that increasing colonic wall thickness probably strengthens the anastomotic fusion. This study is a first step to enlighten the major parameters of tissue fusion, though effects and interactions of various main parameters of bipolar radiofrequency-induced thermofusion of colonic tissue remain unclear. Further studies exploring the main effects and interactions of tissue and process parameters to the quality of the fusion site have to follow. [ABSTRACT FROM AUTHOR]

Organ printing is a variant of the biomedical application of rapid prototyping technology or layer-by-layer additive biofabrication of 3D tissue and organ constructs using self-assembled tissue spheroids as building blocks. Bioengineering of perfusable intraorgan branched vascular trees incorporated into 3D tissue constructs is essential for the survival of bioprinted thick 3D tissues and organs. In order to design the optimal 'blueprint' for digital bioprinting of intraorgan branched vascular trees, the coefficients of tissue retraction associated with post-printing vascular tissue spheroid fusion and remodelling must be determined and incorporated into the original CAD. Using living tissue spheroids assembled into ring-like and tube-like vascular tissue constructs, the coefficient of tissue retraction has been experimentally evaluated. It has been shown that the internal diameter of ring-like and the height of tubular-like tissue constructs are significantly reduced during tissue spheroid fusion. During the tissue fusion process, the individual tissue spheroids also change their shape from ball-like to a conus-like form. A simple formula for the calculation of the necessary number of tissue spheroids for biofabrication of ring-like structures of desirable diameter has been deduced. These data provide sufficient information to design optimal CAD for bioprinted branched vascular trees of desirable final geometry and size. [ABSTRACT FROM AUTHOR]

Abstract: Organ printing can be defined as layer-by-layer additive robotic biofabrication of three-dimensional functional living macrotissues and organ constructs using tissue spheroids as building blocks. The microtissues and tissue spheroids are living materials with certain measurable, evolving and potentially controllable composition, material and biological properties. Closely placed tissue spheroids undergo tissue fusion — a process that represents a fundamental biological and biophysical principle of developmental biology-inspired directed tissue self-assembly. It is possible to engineer small segments of an intraorgan branched vascular tree by using solid and lumenized vascular tissue spheroids. Organ printing could dramatically enhance and transform the field of tissue engineering by enabling large-scale industrial robotic biofabrication of living human organ constructs with “built-in” perfusable intraorgan branched vascular tree. Thus, organ printing is a new emerging enabling technology paradigm which represents a developmental biology-inspired alternative to classic biodegradable solid scaffold-based approaches in tissue engineering. [Copyright &y& Elsevier]

Organ printing or biomedical application of rapid prototyping, also defined as additive layer-by-layer biomanufacturing, is an emerging transforming technology that has potential for surpassing traditional solid scaffold-based tissue engineering. Organ printing has certain advantages: it is an automated approach that offers a pathway for scalable reproducible mass production of tissue engineered products; it allows a precised simultaneous 3D positioning of several cell types; it enables creation tissue with a high level of cell density; it can solve the problem of vascularization in thick tissue constructs; finally, organ printing can be done in situ. The ultimate goal of organ-printing technology is to fabricate 3D vascularized functional living human organs suitable for clinical implantation. The main practical outcomes of organ-printing technology are industrial scalable robotic biofabrication of complex human tissues and organs, automated tissue-based in vitro assays for clinical diagnostics, drug discovery and drug toxicity, and complex in vitro models of human diseases. This article describes conceptual framework and recent developments in organ-printing technology, outlines main technological barriers and challenges, and presents potential future practical applications. [ABSTRACT FROM AUTHOR]

The actual role of programmed cell death (PCD) in embryonic processes and the extrinsic signals that define the death fate in developing cells are still poorly understood. Here, we show that during secondary palate shelf fusion in the mouse, PCD appeared in the medial edge epithelia (MEE) of the anterior region only after shelf contact. Contact was necessary for efficient cell death activation in the MEE. However, exogenous all-trans-retinoic acid (RA) increased cell death independently of contact. Competence to induce cell death by contact or by RA exposure was obtained when the MEE were close to touch. Endogenous RA is a relevant regulator of the secondary palate PCD since this was reduced by a retinol dehydrogenase inhibitor and an RAR specific antagonist. Bmp-7 expression was positively regulated by RA. However, BMP-7 was unable to activate cell death within the palate tissue and NOGGIN, a natural BMP antagonist, did not block PCD. Reduction of PCD at the MEE directly with a caspase inhibitor or by inhibiting retinol dehydrogenase resulted in unfused palate shelves, but adhesion was not affected. In contrast, exogenous RA also blocked fusion, but in this situation the increased cell death within the MEE appeared to affect adhesion, thereby causing cleft palate in vivo. [Copyright &y& Elsevier]

Our previous experimental work showed the feasibility of colon anastomosis using laser; however, it also revealed a narrowing tendency after laser anastomosis. Long-term observation of the postoperative course of laser anastomosis was planned to assess this point.Clinical observation and histologic examination of transverse colotomies (average, 21.2 mm) in New Zealand white rabbits (n=12) closed with a 1,064-nm neodymiumyttrium aluminum garnet (Nd:YAG) laser were carried out up to the third postoperative month, compared with closure of colotomies using a single layer of interrupted sutures of 5-0 Maxon® (Davis & Geck, Pearl River, NY) in a control group (n=12).Whereas the narrowing tendency was observed up to one month after the operation in the laser group (P<0.05), it disappeared at three months following the operation without causing abnormal weight loss during the experiment. Simultaneously, there was again a marked decrease in adhesion formation following laser anastomosis (P<0.01). Collagen fibers were observed earlier in the laser group.An earlier commencement of collagen synthesis after laser anastomosis was proved. A narrowing tendency, which was observed from seven days to one month postoperatively, disappeared at three months after laser anastomosis without exerting any effect on growth. [ABSTRACT FROM AUTHOR]

To assess the possibility of laser anastomosis of the colon, experimental laser closure of colotomies was performed. Transverse colotomies (average 20.7 mm) in New Zealand white rabbits (n = 20) were closed with a 1,064-nm Nd:YAG laser at a pulsating 0.5-W wave of power, using guy sutures to approximate the tissue edges. The laser energy was applied through a specially designed hand piece and hand-held 600- μ m gas-cooled noncontact quartz fiber to produce a satisfactory tissue welding. Similar colotomies were closed using a single layer of interrupted sutures of 5-0 Maxon® (Davis & Geck, Pearl River, NY) in a control group (n=20). Bursting pressure and index of narrowing were recorded immediately after anastomosis and at 1, 4, and 7 days following surgery. Bursting pressure of the laser-welded closure at 1 day was significantly lower than that of the sutured controls (P <0.01); however, all rabbits recovered uneventfully. There was a marked decrease in adhesion formation following laser anastomosis (P < 0.05). Conversely, one control rabbit showed functional stenosis at 4 days. Histologic study revealed an accelerated healing in the laser group. The index of narrowing was significantly higher in the laser group than in the control group (P <0.05). The completely water-sealed laser anastomosis reduces adhesions and is comparable to conventional suture anastomosis of the colon. [ABSTRACT FROM AUTHOR]

A preliminary single-animal study of in vivo argon laser vascular welding was conducted using a canine model. The effects of temperature feedback control and saline drip cooling on patency and collagen cross-linking were investigated. The surface temperature at the centre of the laser spot was monitored using a two-colour infrared thermometer. The surface temperature was limited by either a saline drip or feedback control of the laser. Acute patency was evaluated and collagen cross-link assays were performed. Though both protocols yielded successful tissue fusion, welds maintained at a surface temperature of 50°C using feedback control had an elevated cross-link count compared to controls, whereas tissues irradiated without feedback control experienced a cross-link decrease. Simulations using the LATIS (LAser-TISsue) computer code suggest that drip-cooled procedures achieve significantly higher temperatures beneath the tissue surface than temperature feedback-controlled procedures. Differences between the volumetric heating associated with drip-cooled and feedback-controlled protocols may account for the different effects on collagen cross-links. Covalent mechanisms may play a role in argon laser vascular fusion. [ABSTRACT FROM AUTHOR]