There is a critical need for an ex vivo model representative of the human vascular physiology and encompassing the complexity of the vascular wall and the physical forces regulating its function. Vascular bioreactors for ex vivo culture of large vessels are viable alternatives, but their custom-made design and insufficient characterization often hinders the reproducibility of the experiments. The objective of the study was to design and validate a novel 3D printed cost-efficient and versatile perfusion system, capable of sustaining the viability and functionality of large porcine arteries for 7 days and enabling early post-injury evaluations. MultiJet Fusion 3D printing was used to engineer the EasyFlow insert, converting a conventional 50 ml centrifuge tube into a mini bioreactor. Porcine carotid arteries either left untreated or injured with an angioplasty balloon, were cultured under pulsatile flow for up to 7 days. Pressure, heart rate, medium viscosity and shear conditions were adjusted to resemble arterial in vivo hemodynamics. Tissue viability, cell activation and matrix remodeling were analyzed by immunohistochemistry, and vascular function was monitored by duplex ultrasound. Culture conditions in the EasyFlow bioreactor preserved endothelial coverage and smooth muscle organization and extracellular matrix structure in the vessel wall, as compared to static culture. Injured arteries presented hallmarks of early remodeling, such as intimal denudation, smooth muscle cell disarray and media/adventitia activation in flow culture. Duplex ultrasound confirmed continuous pulsatile blood flow conditions, dose-dependent vasodilator response to nitroglycerin in untreated vessels and impaired dilator response in angioplastied vessels. The scope of this work is to validate a low-cost, robust and reproducible system to explore the culture of native and injured large arteries under pulsatile flow. While the study of vascular pathology is beyond the scope of the present paper, our system enables future investigations and provides a platform to test novel therapies and devices ex vivo, in a patient relevant system.Routine interventions such as balloon angioplasty, result in vascular activation and remodeling, often requiring re-intervention.  Vascular disease is often caused by narrowing or occlusion of blood vessels leading to decreased blood supply to important organs like the heart or brain, or to the extremities. Revascularization procedures are common interventions aimed at re-establishing blood supply to compromised tissues. Typically, a guidewire is passed through an accessible artery to reach the narrowed area and a balloon is deployed to reopen the lumen. While the immediate effect on blood flow is readily achieved, the intervention damages the blood vessel wall which triggers a cascade of acute inflammatory and regenerative responses that result in intimal hyperplasia and vascular remodeling . The vasIn vitro strategies for the study of vascular remodeling focus on 2D culture of vascular cells  or the static culture of rings obtained from blood vessels, the latter importantly incorporates an element of intact extracellular matrix component, which is critical in the remodeling process . Intereysiology –9. This in vivo. We comprehensively characterized the effect of pulsatile flow culture and endovascular injury on the survival, activation and function of vascular resident cells by histology, immunofluorescence and by Doppler ultrasound vasoreactivity measurements.In this study, we developed an open-source 3D printed and economical bioreactor (EasyFlow) which enables the multiplex culture of blood vessels from large animals in small volumes of medium. Furthermore, we have optimized conditions of flow, shear stress, pressure, pulsatility and viscosity to closely mimic the hemodynamic forces applying to an artery ex vivo bioreactor culture for large animal blood vessels, enabling the long-term culture of healthy vessels and the study of pathological mechanisms. Importantly, the open-source nature of our design enables effortless transfer of the system to other labs and reproducibility, promoting the reduction of the use of animals in vascular studies.This work will demonstrate the reproducible application of Carotid arteries were obtained from 4–6 weeks old piglet at The Pirbright Institute, Pirbright, UK. Animal procedures were carried out under the Home Office Animals (Scientific Procedures) Act (1986) and approved by the Animal Welfare and Ethical Review Board (AWERB) of The Pirbright Institute. The animals were housed in accordance with the Code of Practice for the Housing and Care of Animals Bred.Pigs were euthanized by an overdose of 10 ml pentobarbital . All procedures were conducted by Personal License holders who were trained and competent and under the Project License PPL70/8852.Upon exsanguination the neck was opened from the chest cavity to the base of the skull to expose the common carotid artery separating into the right and left carotid artery. Arteries were excised using a no-touch technique to minimize stress to the vascular tissue. Fresh tissue was immediately placed in pre cooled transport media Dulbecco's Modified Eagle's Medium (DMEM) + 20% fetal bovine serum (FBS) + 2% penicillin and streptomycin (P/S) + 1% Amphotericin B (Amp). Tissue was washed two times in transport media and placed on ice for transportation.Following arrival tissue was transferred into laminar hood where further preparation took place. The excess connective tissue was removed in a Petri dish using precision surgical equipment, avoiding any strain to the vessel. Once cleaned, the vessel was placed in new container and washed in transport media (2 × 20 min at 4°C), followed by short term storage in DMEM + 10% FBS + 1% P/S + 1% Amp at 4°C.For each preparation, a sample of 2–4 mm was collected at the time of preparation, as a control.® Fusion 360™ 2.0.5818). Printable file is available in The EasyFlow insert was produced by additive manufacturing which enables for complex design choices to be manufactured with high reproducibility. The CAD model of the system was prepared using Autodesk Fusion 360 , which produced EasyFlow by Polyamide 12 (HP 3D High Reusability PA 12) Black MultiJet Fusion built in 80 μm layers, requiring no additional finish. Considering the constantly expanding list of materials available for manufacturing, the proposed design can be manufactured with alternative materials as long as the additive method resembles a Selective Laser Sintering or Stereolithography type manufacturing.Details of the EasyFlow bioreactor system are presented in The EasyFlow insert consists of a 3D printed part that fits as a lid of a 50 ml standard centrifuge tube. The hexagonal EasyFlow insert presents 6 inlet and outlet ports, creating two separate circulations inside and outside the blood vessel, which is lodged in its adaptor .® Barbed Check Valve, 3/16-inch, WZ-98766-50) added downstream the peristaltic pump, and pressure is stabilized by a pressure dampener system created with a syringe loaded with liquid and air. To increase baseline pressure low bore tubing  was used downstream of the artery. To regulate resistance, an additional clamp  was applied downstream of the resistance tubing. Sterile gas exchange is enabled by the presence of outlets mounted with sterility filters   mounting a 4-cassette head , an EasyFlow insert lodging the blood vessel sample and an equivalent EasyFlow functioning as media reservoir . The two826.001) .The system was monitored by pressure sensors downstream of the sample-containing EasyFlow , and through repeated Duplex ultrasound imaging performed with Logiq V2 .perfusion media (DMEM + 10% FBS + 1% P/S + 1 % Amp + 3% Dextran). The circulation system was inspected for leaks, and any bubble trapped in the system was removed by flowing media across the system.To assure the aseptic environment necessary for the long-term incubation of tissues, all components were sterilized by autoclaving. Elements of the circulation system were assembled under a laminar flow hood and primed with Previously harvested and cleaned tissue was then placed in the EasyFlow unit. Depending on the internal diameter of the vessel segment, an adequate barb connector was selected to facilitate the connection to the perfusion system, typically for porcine carotid arteries 2.4 or 1.6 mm ID connectors were used . The length adaptor within EasyFlow is ultimately adjusted to create a minimal stretch on the longitudinal axis .2 at 37°C). Circulation was connected to the peristaltic pump , the residual air was removed from the newly assembled segment before sealing the system. A sterile syringe containing 3 ml of air and 1 ml of media is connected to the port situated above the artery. This addition serves as a pressure dampener assuring that there is no extreme pressure effecting the artery. Following complete assembly, the system is sealed and transferred it to the incubator (5% COtic pump  and prim® 35 LP Low 4 mm) was inserted into the artery. With a standard in deflator pump the balloon was inflated with phosphate buffer saline (PBS) to 6 atm and the pressure was maintained for 3 min, akin to the procedure performed during peripheral arterial angioplasties. After deflation, the balloon was extracted, the tissue rinsed and placed in the perfusion system for culture, or static culture as a control.A standard peripheral artery angioplasty balloon catheter  in Petri dishes, and incubated at 5% COperfusion media (DMEM + 10% FBS + 1% P/S + 1 % Amp + 3% Dextran). Of note, the addition of dextran exclusively to the flow culture media ensures the appropriate viscosity of the medium which in turn affects the shear stress, while avoiding reducing the diffusion of oxygen in the static cultures, that would represent a limiting factor for the culture. The peristaltic pump is set at the final speed of 14 rpm which, due to the 4 rolls mounted on the peristaltic pump, is equivalent to 56 pulse/min. This pump setting corresponds to 35 ml/min volumetric flow rate . Briefly, ultrasound gel was applied to the outside of the bioreactor to minimize acoustic impedance, compensate for surface incongruencies between probe and bioreactor and allow angulation of probe to obtain an insonation angle of <60°. After clear longitudinal images were obtained, the position of the ultrasound probe and B-Mode settings were adjusted until clear vessel borders with typical M and I lines were visible, and several cine video loops were acquired and saved as video files for off-line analysis. Flow velocity measurements were performed with pulsed wave Doppler. The sample volume was placed at the center of the perfused artery, insonation angle adjusted to <60° and angle correction applied. Image analysis was performed offline with an automated edge detection software . The luminal diameter was measured on video loops over several pulsation cycles. Each diameter measurement represents the average diameter over 0.5–1.0 cm vessel area. The maximal diameter was designated systolic diameter and minimal diameter as diastolic diameter. Volumetric flow rate (Q) was calculated as follows:mean diameter/2)2 and mean diameter = /2.Where In our setup, the mean diameter of the artery was 1.2 mm and a mean velocity of ~50 cm/s  and thisWall shear stress (WSS) was estimated as:*s/cm2 (The viscosity of the perfusion medium (μ) was estimated at 0.035 dyn*s/cm2 . Intimal−9 to 1 M. These concentrations were calculated so that added to 100 ml of medium contained in the bioreactor, they yielded final concentrations ranging from 10−11 and 10−2 M. Nitroglycerin boluses were injected in order of increasing concentration and the reaction of the blood vessel was recorded with a delay of 1 min, once the vessel normalized the next bolus was injected.For the functional vasoreactivity assay, syringes containing 1 ml of nitroglycerin solutions were prepared, with concentration ranging from 10en-face staining or were further processed for histological analysis.Portions of freshly isolated (control) and cultured arteries were fixed in 4% PFA  overnight (O/N) at 4°C. Fixed samples were either used for whole tissue en-face staining was performed on samples washed with PBS and permeabilized with 1v/v% TritonX-100 in PBS for 15 min. Tissues were blocked overnight at 4°C in 20v/v% goat serum in PBS and then incubated with primary antibody solution  overnight followed by goat anti-Rabbit Alexa Fluor 488® (Thermo Fisher Scientific) secondary antibody diluted 1:200 and Phalloidin-iFluor 594 (Abcam- ab176757) for 1 h at 37°C. Nuclei were stained with DAPI  for 10 min at room temperature. Tissues were laid flat and mounted with Fluoromount G . Imaging was performed with Nikon Eclipse Ti A1-A confocal laser scanning microscope, obtaining stack images at 40X along the Z-axis.To establish the maintenance of a continuous endothelial layer facing the lumen and quantify the effect of flow on the alignment of the cells, For histology, tissues were washed with PBS and incubated overnight in 30w/v% sucrose (Sigma-Aldrich) solution in PBS. Following sucrose incubation, samples were embedded in OCT Compound (Agar scientific) in an Iso-Pentane bath cooled using liquid nitrogen. Frozen samples were stored at−80°C until later use. Samples were cryo-sectioned along the transverse plane, obtaining 5 μm thick sections. Sections were collected on microscope slides in quadruplicate, where sections were separated by at least 250 μm.Immunofluorescent staining was performed on prepared sections to quantify luminal coverage, medial integrity and the extent of proliferation. Tissue sections underwent antigen retrieval in a water bath at 80°C for 30 min in tris-EDTA buffer  followed by blocking for 1 h at room temperature with 20v/v% goat serum (Sigma-Aldrich) in PBS. Primary antibodies against CD31 (Abcam-ab28364) diluted 1:200, and Proliferating cell nuclear antigen  diluted 1:100 were incubated overnight at 4°C.® (Thermo Fisher Scientific) secondary antibodies 488 and 567 diluted 1:200 were incubated for 1 h at 37°C. Following secondary antibodies, Human α-Smooth Muscle Actin (SMA) Alexa Fluor® 647-conjugated antibody (R&D Systems- IC1420R) diluted 1:200 was introduced for 1 h at 37°C. Nuclei were stained with DAPI (Merck) for 10 min at room temperature. Incubation with 0.1 w/v % Sudan Black  in 70% ethanol for 10 min at room temperature was performed to reduce tissue autofluorescence. Slides were then mounted in Fluoromount G  and imaged with Nikon Eclipse Ti A1-A confocal laser scanning microscope. Tile images were obtained at 20X to capture the whole tissue section where possible.Appropriate Goat anti-Mouse and Goat anti-Rabbit Alexa Fluor® 6 Vacuum Infiltration Processor). Immunohistochemical assays for Caspase 3 , PDGFR-β  and Vimentin  were similarly outsourced to Veterinary Diagnostic Services, School of Veterinary Medicine, University of Glasgow. Primary antibodies were followed by HRP-conjugated secondary antibody and DAB  staining and counter-staining with hematoxylin. Staining was performed in the Dako Autostainer system. Slices were dehydrated and mounted with Cellpath Mounting Media (SEA-1604-00A).Hematoxylin and Eosin (H&E), and Masson's trichrome staining (MT) were used to estimate the overall maintenance of the tissue structure, at a cellular and extracellular matrix level. These stainings were outsourced to the Veterinary School Pathology Centre at the University of Surrey, and performed by an automated staining system (VIPWhole slice imaging was performed on the NanoZoomer 2.0-HT slide scanner by Hamamatsu.All acquired images were processed in ImageJ v1.53c (FiJi). Custom macros were composed to facilitate image analysis. Where necessary, vessel wall areas  were defined manually and stored as Regions of Interest (ROIs). Resulting ROIs were used to define tissue dimensions and to measure signals specific to the predefined areas.The quantification of PCNA was performed on the fluorescent images collected by confocal microscopy. Tile images covering the cross section of the tissue were obtained to measure distribution of PCNA across the tissue for each section (three sections/sample). PCNA+ nuclei counts were normalized against the total number of DAPI+ nuclei within each area.To define the spatial distribution of PCNA+ cells, images were converted into distance maps where orthogonal distance of each nucleus from the lumen was calculated. Each distance was further normalized against the thickness of the tunica media, to account for differences in tissue morphology between specimens. Individual distance values were organized into a histogram representing the relative frequency of PCNA+ cells against the relative distance from the lumen.SMA intensity in the media was quantified on the immunofluorescence samples by selecting three random, uniformly sized, representative areas within the media of each section. The average signal intensity was quantified in each of the selected areas, and normalized by the total area selected.For histological staining, whole tissue sections were loaded on ImageJ to measure specific signal expression. First an ROI was defined by high-pass gaussian filter to identify the total area of the section. Using a series of user-defined color thresholds, the specific antigen signal was selected and extracted from the image. The area of specific signal was measured and normalized toward the total tissue area.Histological samples prepared with Masson's trichomic staining were used to analyze the coherence of the tissue. Three random, uniformly sized, representative regions were selected to perform the analysis. In each region, the area occupied by the tissue vs. the empty spaces were measured and normalized toward the total area of measurement.The quantification of endothelial coverage was performed on the fluorescent images obtained by confocal microscopy. To quantify the coverage, the fraction of CD31-expressing lumen over the total length of the portion of lumen perimeter captured in each picture was calculated in vessel cross-sections.en-face confocal images compressed into a Z-Max projection. In each picture, three random areas were selected and a vector map of the Phalloidin signal was generated using OrientationJ (ImageJ/FiJi plugin) to obtain the local orientation of the signal within the selected area. Resulting vector values were organized into a histogram to represent the relative frequency of angular orientations, where zero is the representative value of fibers perpendicular to the flow direction.Fiber orientation was quantified in the Experiments were repeated in 3 to 5 biological replicates. In some analyses, individual samples have been excluded due to poor quality of the preparation or staining.post-hoc test (GraphPad Prism 8.1.2). A value of p < 0.05 was considered as statistically significant. Data are presented as mean ± SD.Difference among groups were evaluated using one-way ANOVA or Kruskal–Wallis test, based on results from normality tests, followed by Fisher's LSD The resolution of MultiJet fusion 3D printing enabled the efficient and cost-effective production of the EasyFlow insert, incorporating detailed features, and producing a non-porous surface. The resulting product is biocompatible and autoclavable multiple times. The insert is designed to fit as the lid of a 50 ml centrifuge tube, creating a versatile and self-contained bioreactor for vascular culture . The EasThe EasyFlow system was created as a self-contained, off-the-shelf solution for a wide range of experimental setups, and therefore was designed to maximize the included features, while maintaining a compact design. The hexagonal shape provides ideal internal space to position the necessary features, and enables to spatially distribute the connectors externally, allowing easy handling and setup, and offering enough space flexibility to connect external components.*cm2 or 3.5 cP).The blood vessel is accommodated in an adjustable adaptor, through barb connectors of interchangeable size . Once then face staining, immunofluorescence imaging and histology in porcine carotid artery segments cultured in EasyFlow or in static culture for 7 days, and compared them to their freshly isolated segments that were used as “untreated” controls.Next, we tested how the EasyFlow environment impacted on cellular physiology and ultrastructural components, by performing Hematoxylin and eosin staining revealed that the overall structure of the tissue was preserved during culture under flow conditions. In particular, the vessel wall layers appear unaltered, and no remodeling was observed . Some ofExtracellular matrix provides the mechanical support and elasticity necessary for blood vessels to perform their function, cell apoptosis and abnormal activation may affect this structure, weakening the vessel wall. We visualized the collagen content by Masson's trichrome staining and confirmed the maintenance of the extracellular matrix organization in tissues cultured in EasyFlow, while static culture produced a visible loss of tissue integrity . These oImmunofluorescence staining revealed the finer changes in cell distribution. Both endothelial coverage of the lumen and expression of smooth muscle actin in the media were slightly reduced in the static condition, as compared to the flow culture, although not to a significant level . These men face staining was performed to better visualize endothelial coverage and media organization. Overall, en face staining revealed a good luminal coverage in both culture conditions  and 1.19 ± 0.30 mm (“diastolic”). The ‘peak systolic velocity' was 52 ± 13 cm/s and ‘mean velocity' was 44 ± 8 cm/s, resulting in an estimated volumetric luminal flow of 33 ± 16 ml/min . While control retained the diameter at day 7, injured arteries significantly decreased diastolic diameter . There were no significant changes in luminal flow (p = 0.355), it was decreased as compared to control in angioplastied arteries at day 3 (0.15 ± 0.01) and increased at day 7 (0.38 ± 0.06 mm)  . We also0.06 mm) .en face staining. As expected, balloon injury determined a significant loss of cells and disorganization in the extracellular matrix in the intima and the medial layers, as shown in the H&E and Masson's trichrome staining ] and hypercholesterolemia   . These iDespite these limitations, these models delivered great insights in the understanding of the mechanisms of vascular homeostasis and the patterns associated with common diseases, such as atherosclerosis and restenosis . The strVascular bioreactors have been developed both at commercial level and in-house to provide a suitable physiological environment to maintain blood vessels in culture or enhance the maturation of tissue engineered constructs.In this study, we present a 3D printed bioreactor that enables the culture of blood vessels within a 50 ml centrifuge tube, enabling small volume cultures and multiplexing. Previous work has demonstrated the feasibility and practicality of a bioreactor designed to fit as a lid of a 50 ml tube, however our system is distinctive as the whole insert is created entirely by 3D printing , 16.EasyFlow's open-access design allows simple uptake by other labs, and the adjustable vessel compartment enables customization to different vessel sizes and tissue sources by simply exchanging the barbed connectors and adjusting the length adapters.in vivo environment experienced by the blood vessel in its natural location. Pump revolution rate was adjusted to impose a “heart rate” of 56 beats/min to simulate resting heart rate and continue pressure monitoring confirmed a physiological range of 60–120 mmHg, which is compatible to the average healthy blood pressure in the human population. Common carotid arteries experience a mean flow velocity of 15–43 cm/s (end diastolic-peak systolic) and a wall shear stress in the range of 6–21 dyne/cm2 , this parameter closely affects the shear stress experienced by vascular cells in flow, according to the Hagen-Poiseuille equation .Our in-depth consideration and characterization of the conditions and forces acting on the vessel in culture is uncommon in previously published studies , 19–21. When compared to static culture, EasyFlow cultured arteries demonstrated an overall better maintenance of tissue integrity and structure. In particular, the organization of the smooth muscle cells and the extracellular matrix were closer to the native tissue. Interestingly, we did not detect a critical difference in lumen coverage, apoptosis or activation of the vessel wall in the two culture systems. This is not completely unexpected as the static culture was designed to replicate the vessel ring culture, with tissue specimens of 2–5 mm in length –25. PrevAfter verifying the feasibility of a pulsatile culture using EasyFlow, we assessed the effect of injury in the flow cultures. We delivered the injury before setting up the static or flow culture, but it is important to note that, thanks to the multiple access ports available on EasyFlow, the catheter-mediated balloon injury could potentially be implemented on the mounted sample at any point of the culture, without disturbing the system.In vivo, mechanical damage and flow disruption, accompanied by damage to the medial layer , results in smooth muscle cells activation, proliferation and migration into the intima layer, contributing to the thickening of the vessel wall and effectively narrowing the lumen (restenosis) (tenosis) , 27. Advtenosis) , 29.Following balloon application and flow culture, we observed significant damage to the intima and media layers of the tissue, which is comparable to what reported in similar animal models where intima denudation and early media apoptosis drive the regeneration process . In addiin vivo after at least 2 weeks in rabbits, a typical model for vascular remodeling .All raw data, images, and analyses supporting the findings of this study are available through the online repository Zenodo  of the Pirbright Institute. All procedures were conducted by Personal License holders who were trained and competent and under the Project License PPL70/8852.RM conceived, planned the experiments, and analyzed the data with contribution by CH. DM and CH provided key experimental expertise. RM and PC wrote the manuscript and prepared the figures with support of CH. PC, CH, and JM conceived the original idea and supervised the project. All authors contributed to the article and approved the submitted version.RM was supported by the Doctoral College studentship award (University of Surrey). DM and PC were supported by the National Center for the Replacement, Refinement & Reduction of Animals in Research (grant numbers: NC/R001006/1 and NC/T001216/1).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


ELT) and fractal dimension (D) values (P < 0.05), and the stability of soil structure and the capacity of soil moisture retention has been significantly improved. The SOM content had a significant positive correlation with MWD, GMD, and > 2 mm water-stable aggregates and a significant negative correlation with the ELT, D, and water-stable microaggregates. In particular, the organic–inorganic coupling treatment of TFO showed the highest SOM content, soil moisture content, water-stable macroaggregates and maize yield, which was the most appropriate amendment for improving the reclaimed soil structure and fertility of Hollow Village in Loess Area.In order to solve the soil problem of poor structure and low fertility after the abandoned homestead reclamation of Hollow Village in Loess Area and to improve the quality of the reclaimed soil in Hollow Village, a five-year field experiment was conducted here. In this experiment, the following seven treatments were applied: no modified material (CK), maturing agent (TM), fly ash (TF), organic fertilizer (TO), maturing agent + fly ash (TMF), maturing agent + organic fertilizer (TMO) and fly ash + organic fertilizer (TFO), and the effects of different improved materials on soil properties and crop yield were studied. The results showed that: soil organic matter (SOM) and total nitrogen contents increased significantly after the application of different improved materials, which promoted the cementation and aggregation of water-stable microaggregates (< 0.25 mm), and the water-stable macroaggregates showed an increasing trend. In the 0–0.15 m soil layer, the proportion of water-stable macroaggregates under TM, TF, TO, TMF, TMO, and TFO treatment increased by 328.2%, 130.0%, 87.8%, 81.1%, 36.7%, and 12.2% compared with CK, respectively. Meanwhile, TF, TO, TMF, TMO, TFO treatments significantly increased the mean weight diameter (MWD) and geometric mean diameter (GMD) values, reduced soil bulk density, the stable aggregate index ( Therefore, a great deal of abandoned housing resources is left idle and wasted3. Moreover, the rural housing land construction generally shows an undesirable trend of "building the new but not demolishing the old", occupying high-quality arable land to expand to the periphery, resulting in the destruction and occupation of a large number of arable land resources5. Rural hollowing seriously threatens arable land resources and regional food security, and has become a major bottleneck limiting the construction of villages and the coordinated development of urban and rural areas6. In view of the above problems, combined with the rising contradiction between human and land in the loess plateau region and the increasing local demand for reclaiming the abandoned housing land, it is necessary to promote comprehensive land improvement in hollow villages and carry out reclamation of abandoned housing land for implementing "requisition-compensation balance" of arable land and alleviating the regional contradiction between human and land8. However, hollow village reclaimed soil mainly comes from the old wall soil on the abandoned housing land, which is mostly raw soil that has not been cultivated for years due to the combined influence of natural and human factors, and has lost its natural functions and properties. Its physical structure and soil fertility are seriously damaged, which limits the land productivity and sustainable development of new arable land in hollow villages. It is necessary to improve the structural properties and fertility features of the reclaimed soil on the housing land and to improve the productivity of the reclaimed soil10.At present, the loess plateau region is facing the problems of fragile ecological conditions, decreasing arable land area, lack of arable land reserve resources, and intensifying human-land conflicts. With the rapid promotion and implementation of urbanization and industrialization, a large number of rural laborers migrate to cities, resulting in the continuous deterioration of abandoned and hollowed rural areas13. Compared with non-water-stable aggregates, water-stable aggregates play a more important role in maintaining soil structural stability and erosion resistance, and retaining soil nutrients15. It was found that the increase of organic matter content is directly related to the increase of the number and stability of water-stable aggregates, and the application of different improved materials can effectively increase the organic matter content of the soil, promote the cemented agglomeration and structural stability of soil aggregates, and improve the fertility and erosion resistance of reclaimed soil17. Fly ash is the powdery solid residue discharged from coal-fired power plant after the combustion of pulverized coal, which contained abundant clay particles and oxides such as Al2O3 and Fe2O3, it could significantly improve mutual adsorption and aggregation between soil particles and enhance the soil water and fertility retention19. The maturing agent of ferrous sulfate can improve soil structure and reduce the pH value of soil while loosening the soil, it can also be used as fertilizer, which plays an important role in the absorption of plants20. Organic fertilizers are rich in organic matter and various nutrients, which can effectively improve soil fertility and structure, increase soil productivity and maintain the sustainability of crop yield22. Therefore, it is of practical importance and necessity to enhance the structural stability and fertility feature of the reclaimed soil by applying different soil improved materials to mature and improve the reclaimed soil of hollow villages, so that the reclaimed soil can recover its original functions and properties. The current approaches to remediation of hollow villages are mainly focused on the assessment of remediation potential, evolution laws, regional macro-remediation models and regulation policies, which further affirm the significance of the remediation of abandoned housing land in hollow villages25. However, there are few studies on the effects of different improved materials on hollow village reclaimed soil, which seriously restrict the availability and sustainable development of hollow village reclaimed soil.The number and distribution ratio of soil aggregates are not only important indicators of soil erosion, hardening, compaction and other structural conditions, but also play a key role in supplying and storing soil nutrients, regulating soil water holding capacity and maintaining land productivity, and they are key indicators that can well evaluate the changes in soil fertility feature and environmental quality1The reclaimed soil in hollow villages is taken as a key initiative to the regional supplementation to arable land resources; however, little research is conducted to compare and analyze the role of different improved materials in the structure and fertility improvement of reclaimed soil, thus it is difficult to distinguish the differences in influences of different improved materials on the number of aggregates, structural stability, nutrient content and crop yield of reclaimed soils. In order to make up for the lack of information about the effect of different improved materials on the hollow village reclaimed soil, the objectives of this paper were to evaluate : (1) the effects of different improved materials on soil nutrient content, aggregate distribution and stability, soil bulk density and soil moisture content; (2) the correlation between the soil organic matter and the water-stable aggregates distribution and stability indexes; and (3) the effects of different improved materials on maize yield in hollow village reclaimed soil. The research results will provide a theoretical basis for selecting appropriate amendment materials to improve the structure and quality of reclaimed soil in the hollow village.P < 0.05), among which TFO treatment was the most effective on improvement of the organic matter content. In the 0.15–0.30 m layer, the results of significance analysis showed that TO, TMF, TMO and TFO treatments all significantly increased the organic matter content (P < 0.05), while TM and TF treatments had no significant difference in improving the soil organic matter content, with TFO treatment having the most significant effect.After the application of different improved materials, the SOM and TN contents in both 0–0.15 m and 0.15–0.30 m layers of the hollow village reclaimed soil showed an overall increasing trend . In the 0–0.15 m layer, after the application of different improved materials in hollow village reclaimed soil, the proportion of water-stable macroaggregates (particle size > 0.25 mm) showed an overall increasing trend, and the water-stable microaggregates content (particle size < 0.25 mm) showed a decreasing trend. In particular, it showed that except for the treatment of maturing agent (TM), the proportion of water-stable aggregates  were significantly increased under the TF, TO, TMF, TMO and TFO compared with CK, especially that of particle size > 2 mm  , and fractal dimension (D) are important indicators reflecting the structural geometry and stability of soil aggregates, and it has been indicated in this research that the higher the MWD and GMD and the smaller the ELT and D, the better the structural stability of the aggregates and the soil structure28. Compared with CK treatment, the MWD and GMD showed a trend of significant increase while the D and ELT showed a trend of significant decrease (P < 0.05) under TF, TO, TMF, TMO and TFO treatments after the application of different improved materials, and TM treatment had no significant effect on the indicators of aggregate stability . It can be seen from the data of aggregate stability indicators that the structural stability of water-stable aggregates in the 0.15–0.30 m layer shows a decreasing trend compared with the 0–0.15 m layer, which may be related to the higher organic content in the 0–0.15 m layer.The mean weight diameter (MWD), geometric mean diameter (GMD), unstable aggregate index (ty Table . In the P < 0.05)  is one of the important indicators reflecting soil quality, and the BD at the 0–0.15 m and 0.15–0.30 m layers of the reclaimed soil decreased significantly after the application of different improved materials (05) Fig. a. In theP < 0.05), and the variation of SMC in the two soil layers under different treatments was basically similar, showing as follows: TFO > TMO > TMF > TO > TF≈TM > CK  of the reclaimed soil in the 0–0.15 m and 0.15–0.30 m layers increased significantly after the application of different improved materials , indicating that soil organic matter was an important factor affecting the formation of water-stable aggregates and their structural stability., and higher organic matter content would promote the formation of macro water-stable aggregates and improve the structural stability of soil. The water-stable aggregates  content had a significant positive correlation with MWD and GMD values and a highly significant negative correlation with ELT and D values; water-stable microaggregates (< 0.25 mm) had a highly significant negative correlation with MWD and GMD values and a significant positive correlation with D, indicating that the increase of water-stable aggregates with larger particle size helped to promote the structural stability of soil aggregates. In summary, it showed that TM, TF, TO, TMF, TMO and TFO treatments of improved materials can effectively promote the formation of macro water-stable aggregates and improve their structural stability while promoting the increase of organic matter content in the hollow village reclaimed soil. In particular, TFO treatment was more beneficial to improve the structural properties of hollow village reclaimed soil, enrich the soil fertility, and enhance the erosion resistance.To further explore the correlation between the parameters of the reclaimed soil after the application of six different improved materials, a regression analysis was conducted in this paper on the correlation between the parameters of organic matter and water-stable aggregates with different particle sizes. From Table P < 0.05). The average kernels per ear and 100-kernel weight under different treatments showed a similar increasing trend to the maize yield, highest under TFO, following by TMO and TO. Compared with CK, the 100-grain weight increased by 2.0%, 3.9%, 8.1%, 4.8%, 4.9% and 12.5% respectively under TM, TF, TO, TMF, TMO and TFO treatments, and the maize yield increased by 10.1%, 18.2%, 34.1%, 24.9%, 38.8% and 53.4%, respectively. The maize yield under TFO was the highest, up to 11,558.79 kg ha−1. In summary, it showed that organic improved materials had better effect on improving the maize yield than inorganic improved materials. The organic–inorganic coupling treatment of TFO and TMO had the best effect on improving the 100-grain weight and maize yield of the reclaimed soil. The possible reason was that the combination of organic and inorganic constituents can effectively increase the soil organic matter and total nitrogen contents, promote the formation and cementation of aggregates, increase the retention and transmission of water and improve the structural stability of hollow village reclaimed soil, which were confirmed by the results of the previous effects on SOM, total nitrogen, soil moisture content, aggregate proportion and aggregates structural stability index.Different improved materials showed a different effect on maize yield Table . In part30. Improved materials such as organic fertilizer, maturing agent (ferrous sulfate) and fly ash can effectively improve soil organic matter content when applied alone or returned to the field after coupling. The cementing substances formed during conversion and decomposition contribute to the cementation and aggregation of soil aggregates and have significant effects on the size distribution and structural stability of the aggregates31. In this study, except for TM treatment, TF, TO, TMF, TMO and TFO all significantly increased the > 2 mm and 1–2 mm water-stable aggregates content and decreased water-stable microaggregates content (P < 0.05) compared with CK. In particular, the treatment of fly ash + organic fertilizer (TFO) had more positive effects on the water-stable macroaggregates content, which may be partially attributed to the higher SOM content under the TFO treatment. Our results are partially consistent with that of Singh et al. and Zhang et al., who found that the combined application of organic and inorganic improved materials showed higher SOM content than the application of either amendment material alone, and therefore had a more positive effect on promoting the formation and preservation of macroaggregates33.Soil organic matter, oxides and clay minerals are the main cementing substances for the formation and stability of soil aggregates, and play important roles in the content, size distribution, and structural stability of soil aggregates. showed that, as fly ash had already been recognized as a potential soil amendment for improving soil physicochemical properties, it could significantly improve the mutual adsorption and aggregation between soil particles after being applied to soil34. The maturing agent of ferrous sulfate could effectively improve soil structure and reduce the pH value of soil while loosening the soil35. The cementing substances such as polysaccharide and humus generated by the decomposition after applying organic fertilizer to soil could further promote the aggregation of soil particles and the stacking and cementation of micro aggregates to macroaggregates, increasing the water-stable macroaggregates content and soil structural stability36. Therefore, the organic–inorganic coupling treatment of fly ash + organic fertilizer can effectively increase the organic matter content of soil. In addition, fly ash has well-developed specific surface area and multi-level pores, and contains abundant clay particles and oxides, therefore it can significantly promote the formation and stabilization of the aggregates in reclaimed soil. This is similar to the stability indicators of aggregates in this study that MWD and GMD values showed a significant increase under TF, TO, TMF, TMO and TFO treatments while D and ELT values showed a significant decrease (P < 0.05). Among six amendment material treatments, the indicator of aggregate stability under TFO treatment is the highest, so the coupling treatment of fly ash + organic fertilizer (TFO) has a more effective effect on improving the structural stability of hollow village reclaimed soil aggregates and may be considered as an appropriate way to increase the content and structural stability of reclaimed soil aggregates. Moreover, the coupling treatment of TFO can also significantly reduce soil bulk density and increase total nitrogen and soil moisture contents compared with other treatments, which was helpful to promote the growth and yield of maize crops. The research result is similar to the present findings of Chang et al. and Wei et al., who indicated that the combined application of organic and inorganic modified materials were more conducive to the improvement of soil quality and crop yield than the application of either amendment alone38.The Study by Blissett et al39. Reasonable conversion of waste resources into useful and sustainable materials can not only increase the soil organic matter content, but also improve soil fertility, promote plant growth and contribute to the sustainable development of agriculture40. Fly ash is waste for power plants, which, however, was very useful as a low-cost amendment for soil structure and nutrient improvement41. Considering the increasing cost of chemical fertilizers, Ren et al. and Singh et al. found that, within a certain range, the combined application of organic and inorganic materials could better improve the reclaimed soil structure and fertility with less cost, which was easily accepted by farmers42. Therefore, the combined treatment of organic fertilizer and fly ash (TFO) can not only meet the investment requirements in land reclamation, but also effectively improve soil fertility, and has a grear potential for application and promotion in land reclamation43. However, the application of inorganic modified materials alone was not conducive to the adjustment and restoration of reclaimed soil physical structure or the correction of soil nutrient imbalance, which affected the improvement of soil organic matter and microbial activity22. In conclusion, the preference that we recommend for the application of modified materials is as follows: fly ash + organic fertilizer (TFO) > maturing agent + organic fertilizer (TMO) > organic fertilizer (TO) > maturing agent + fly ash (TMF) > fly ash (TF) > maturing agent (TM).Organic fertilizer and fly ash are commonly used local waste resources, and their unreasonable utilization will lead to serious resources waste and environmental problems31. In this study, after the application of different improved materials, the organic matter and total nitrogen contents in both 0–0.15 m and 0.15–0.30 m layers of the reclaimed soil showed a trend of overall increase. In the 0–0.15 m layer, the organic matter content increased by 9.6%, 79.0%, 90.0%, 61.4%, 120.1% and 131.7% respectively under TM, TF, TO, TMF, TMO, and TFO treatments compared with CK treatment, indicating that different improved materials all played important roles in improving the organic matter content of reclaimed soil. However, the improvement effect on the organic matter and total nitrogen contents of reclaimed soil differed due to the different physicochemical properties of different improved materials and the difference in the process of influencing the microbial activity in reclaimed soil. In particular, fly ash + organic fertilizer (TFO) had the best improvement effect, followed by maturing agent + organic fertilizer (TMO) and organic fertilizer (TO), which was directly related to the nutrient content and structural condition of fly ash and organic fertilizer. Fly ash is beneficial to soil microbial activity and nutrient decomposition, and endows reclaimed soil with the property of good fertilizer retention19. In addition, organic fertilizers are rich in organic substances and various nutrients, so the organic–inorganic coupling of fly ash + organic fertilizer (TFO) has the most significant effect on the improvement of organic matter. The research of Zhang Xuhui et al. and Six et al.45 showed that the accumulation of organic matter is closely related to 0.25–2 mm aggregates content, and the larger the particle size, the higher the organic carbon content. TFO treatment significantly increased 0.25–2 mm aggregates content, which also had an important contribution to the improvement of soil organic matter content. This was similar to the findings of Lei et al.42 which showed that the compound returning of the organic fertilizer of rotted chicken manure and the inorganic improved material of fly ash could significantly increase organic matter content.The application of different improved materials is the important source for the enhancement of organic matter and total nitrogen in reclaimed soil. Soil improvement materials such as organic fertilizers, maturing agents (ferrous sulfate) and fly ash, when applied alone or returned to the field after coupling, can enhance the fertility of the soil, promote the increase of crop biomass, and increase the amount of plant residues and roots returned into the soil, thus increasing the soil organic matter contentELT, D and micro water-stable aggregates content (particle size < 0.25 mm). The proportion of water-stable aggregates  was significantly correlated with the values of MWD and GMD, indicators of aggregate stability, indicating that soil organic matter content affected the cementation, aggregation and structural stability of soil aggregates47. The returning and application of exogenous improved materials promoted the increase of organic matter content in hollow village reclaimed soil, which created good conditions for the cementation and aggregation of small and medium-sized soil particles and helped micro water-stable aggregates form into macro water-stable aggregates through the adhesion and aggregation of organic matter, thus enhancing the structural stability and erosion resistance of soil.The results of the correlation analysis between the parameters of hollow village reclaimed soil showed that the soil organic content had a highly significant positive correlation with MWD, GMD, and water-stable aggregates (particle size > 2 mm) and a highly significant negative correlation with D and ELT, indicators of aggregate structural stability, showed an overall increase, and the content and structural stability of water-stable macroaggregates in reclaimed soil were enhanced. Among the six different improved material treatments, the organic-inorganic coupling treatment of fly ash+organic fertilizer (TFO) was the most suitable way for the improvement of hollow village reclaimed soil, and it can effectively increase soil organic matter, total nitrogen, soil moisture and water-stable macroaggregates contents and enhance the structural stability and erosion resistance of the hollow village reclaimed soil, thereby improving the soil fertility.After the application of different improved materials, the soil organic matter and total nitrogen contents in hollow village reclaimed soil showed an increasing trend, among which the coupling treatment of fly ash+organic fertilizer (TFO) had the best effect on the enhancement of the organic matter and total nitrogen contents in the 0–0.15 m soil layer, with an increase of 131.7% and 51.02% respectively. Meanwhile, the various improved materials significantly increased soil moisture content and reduced soil bulk density. With the increase of organic content, different amendment materials promoted the cementation and aggregation of water-stable microaggregates to water-stable macroaggregates (>0.25 mm). The water-stable macroaggregates content in the 0–0.15 m layer under different treatments showed as follows: TFO>TMO>TO>TMF>TF> TM>CK, and the largest increase was shown under fly ash+organic fertilizer treatment (TFO), being 328.2%. The values of MWD, GMD, Based on the Key Laboratory of Degraded and Unused Land Remediation Project, Ministry of Natural Resources, the long-term positioning test plot for reclaimed soil improvement of hollow villages was set up in the pilot base of Fuping County, Shaanxi Province, China  Fig. . The stu−3, the soil texture was silt loam (US Soil Taxonomy) with 10.15% clay (particle size < 0.002 mm), 77.82% silt (particle size of 0.05–0.002 mm), 12.65% sand (0.05–2 mm), 4.4 g kg−1 organic matter, 0.15 g kg−1 total nitrogen, 61.3 mg kg−1 available potassium and 70.4 mg kg−1 effective phosphorus. The proportion of water-stable aggregates with particle size > 2 mm, 1 ~ 2 mm, 0.5 ~ 1 mm, 0.25 ~ 0.5 mm and < 0.25 mm are 0.67%, 0.84%, 1.67%, 3.55%, 93.27%, respectively. In summary, the reclaimed soil of the housing land in hollow villages was mainly developed from loess parent material, and the soil fertility and structure were comparatively poor.The basic physicochemical indicators of the reclaimed soil at the depth of 0 to 0.30 m before the the experiment were as follows: the pH value (water-soil mass ratio 1:2.5 ) was 8.5, the soil bulk density was 1.40 g cm4) were selected as the improved materials for reclaimed soil  and ferrous sulfate FeSO were sel49, soil bulk density and water content were determined by cutting ring method and drying method at 105 °C50, and the distribution and size of water-stable aggregates in two soil layers at the depth of 0–0.15 m and 0.15–0.30 m were determined by the wet-sieve method44. See Eqs. (ELT), and Fractal Dimension (D)53. Maize yield was determined by random sampling method, and the maize in the 21 experimental plots with an area of about 84 square meters were manually harvested at maturity. Ten representative plants were randomly selected for each treatment at maize maturity, and the effective panicle number and number of kernels per spike were counted. The maize kernels were dried at 80 °C for 48 h to ensure that the moisture content was kept below 15%, 100-kernel weight of maize was measured by electronic balance, and the theoretical yield of maize was calculated54.ith sieve, Wi is the weight of aggregates retained in the ith sieve, M(r ≤ xi) is the weight of aggregates with a fraction diameter less than or equal to xi, MT is the gross weight of aggregates, and R0.25 is macroaggregates with diameters of > 0.25 mm.The soil samples were tested in late September 2020 after the winter wheat harvest, and the undisturbed soil samples were collected for determining soil aggregates and the mixed soil samples for determining the organic content from the soil layers at the depth of 0 to 0.15 m and 0.15 to 0.30 m respectively. Every treatment was sampled three times repeatedly. The aggregate soil samples were preserved in stainless steel aluminum boxes, and the influence on the structure of soil aggregates should be avoided as much as possible during transportation. After being brought back to the laboratory, the undisturbed soil samples were air-dried and gently broken into small pieces of about 10 mm according to the natural cracks, and impurities such as stones were removed for the testing of water-stable aggregates, organic matter and other indicators. The soil organic matter and total nitrogen contents were determined by the potassium dichromate—external heating method and Kjeldahl methodhttps://www.microsof.com/zh-cn/download/ofce.aspx) and SPSS25.0 . Differences among treatments were evaluated by one-way analysis of variance (ANOVA), the least significant difference (LSD) method was used for testing the significance of differences among different treatments (P < 0.05), and all data are normally distributed or close to normal distribution before ANOVA.Data statistics and analysis were performed by Microsoft Excel 2010  cultivar that we used in the present experiment, complied with institutional, national, international guidelines. We complied with the IUCN Policy Statement on Research Involving Species at risk of extinction and the Convention on International Trade in Endangered Species of Wild Fauna and Flora.“Xianyu 335”, the maize (


Spent broiler hen meat is sold at a lower price, owing to its poor texture and lower acceptability, in comparison with broiler meat. The tenderization of spent hen meat using kiwi and pineapple extracts will encourage meat processors to use this less expensive meat as a new source of raw materials for the production of different chicken meat-derived products, particularly when solving the problem associated with a great shortage of raw chicken meat materials. Therefore, the present study was undertaken to investigate the effect of kiwi extracts (5 and 7%), pineapple extracts (5 and 7%), and a combination between the two (5% kiwi and 5% pineapple) on the structural changes, electrophoretic pattern, and quality attributes of spent hen meat patties. The results demonstrated that all extract-treated meat patties exhibited a significant decrease in collagen content and shear force value, a significant increase in collagen solubility percentage, and significant improvements to all sensory attributes, in contrast to their counterpart control samples. Additionally, a non-significant change in lightness and yellowness values and a significant increase in redness value were observed in all extract-treated specimens. Moreover, the treatment of spent hen meat patties with kiwi and pineapple extracts resulted in marked degenerative changes of the muscle fiber and connective tissue, as well as a decrease in protein bands, with subsequent enhancement in tenderness. The effect was more highlighted in specimens treated with pineapple extracts (7%) and with kiwi (5%) and pineapple extracts mixture (5%). The tremendous growth of the poultry industry led to a wider availability of meat layers at the end of their economically productive life. Spent broiler hens produce heavy broiler birds with an adequate amount of breast and thigh meats . MoreoveThe disposal of unutilized spent broiler hens is considered one of the main environmental and economical burdens of the poultry industry. In this sense, the development of comminuted meat products from such meat offers an important avenue for their profitable disposal. Therefore, the production of comminuted meat products, as well as the commercialization of unconventional, less valuable spent broiler hen meat should be highlighted due to the increase in broiler meat availability . Among tAttempts of processing meat products from spent hen meat have been reported by Indumathi et al. . HoweverThe tenderization of spent hen meat using natural sources, such as fruits, will encourage the meat processors to use this meat as a new source of materials for manufacturing of various chicken meat products, especially to solve the problem associated with a great shortage of raw chicken meat materials. The pineapple fruit has excellent juiciness and flavoring properties and can be used as a tenderizing substance in meat due to its bromelain content . LikewisMost of the previous studies focused on using kiwi and pineapple extracts for the tenderization of spent hen meat ,14. NoneA three replicate-based experiment (three samples per replicate) was performed to evaluate the effect of kiwi extracts (5 and 7%), pineapple extracts (5 and 7%), and their combination (5% kiwi and 5% pineapple) on the structural modifications, electrophoretic pattern, and quality properties of the product during the formulation of spent hen meat patties.Fresh pineapple and kiwi fruits were purchased from a local supermarket in Cairo, Egypt. Each fruit was de-peeled, chopped into slices, and blended with an equal volume of chilled distilled water, for 1–2 min. The resulting specimen of each fruit was then filtered through four layers of muslin cloth and the filtrate was recovered as raw fruit extract. This crude extract was used as a potential source of proteolytic enzymes for further application in meat .3) of about 100 g. Sodium tripolyphosphate and seasonings mix were acquired from Loba Chemie, Mumbai, India while the sodium chloride and starch were purchased from a local market in Cairo, Egypt.Ten dressed carcasses of spent hen (4 kg each) were purchased for each replicate from a local spent hen plant in Cairo, Egypt. The carcasses were transferred to the lab and stored overnight at 4 °C, for 24 h. The breast and thigh muscles were then excised from the carcasses and trimmed of obvious fat and connective tissue, then cut into small cubes  from each group, as well as the control, non-marinated meat cubes, were ground with a plate grinder , and then blended with water (18%), salt (1.5%), starch (5%), polyphosphates (0.3%), and seasonings (0.2%). Each mixture was shaped into burger patties (about 80 g and 1.5 cm thick) by means of a standard burger maker (9 cm inside diameter), embedded in plastic wrapping films, kept at −40 °C for 35 min, placed in plastic recipients, and then stored at −18 °C for 3 months. For each replicate, samples were taken from each group for 2nd day (zero time) and monthly examinations.Collagen content and collagen solubility% of spent hen meat patties from each group were analyzed in agreement with the method described by Naewbanij et al. .The shear force of spent hen meat patties from each group was determined using the method recommended by Shackelford et al. , by mean65, the aperture size of 11 mm, an observer of 10°, and calibrated against a white calibration plate, using the method outlined by Shin et al. [The color values of spent hen meat patties from each group were measured by using a Chroma Meter , with an illuminant Dn et al. . From eaSensory characteristics of the spent hen meat patties samples were analyzed following the AMSA guidelines . Twenty-Spent hen meat patties samples (1 × 1 cm) from each group were fixed for 24 h in 10% formalin and prepared and stained with Hematoxylin and Eosin, using the method described by Banchroft et al. . At leasSpent hen meat patties samples were sliced into small parts (2 × 2 × 3 mm) and directly fixed in 2.5% phosphate-buffered glutaraldehyde, at 4 °C, for 2 h. Each sample was washed using phosphate-buffered saline (0.1 M), three times (30 min per time), then dehydrated in ethanol , followed by drying for 15 min, using a critical point drier . Finally, samples were coated with gold in a vacuum evaporator  and were inspected for their topographical structure, using a scanning electron microscope  .g and the supernatant was dialyzed against sodium phosphate buffer (0.1 N) containing 0.1% 2-mercaptoethanol. Dialyzed solution (50 µm) was taken for loading the gel and electrophoresis was conducted at constant voltage mode (100 V/slab and 30 mA) for 5–6 h until the dye reached the lower point of the gel. After that, the gel was removed and subsequently stained with Coomassie blue (4–5 h), destained, and then photographed.Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) was performed using an Omni PAGE Maxi, Stock (VS20-48-1)  electrophoresis system . Five grp < 0.05 levels.The obtained results were statistically analyzed using the SPSS statistics 17.0 for windows . The one-way analysis of variance (ANOVA) test was used in order to compare the data and the least square difference (LSD) test was applied to determine the significance, since the differences were considered significant at p < 0.05) reduction in the collagen content and shear force value, along with a significant (p < 0.05) increase in the collagen solubility %, as compared to their counterpart control samples , pineapple (5 and 7%), and a mixture of kiwi (5%) and pineapple (5%) induces a significant  change in lightness (L*), as well as yellowness (b*) values, with a significant (p < 0.05) increase in redness (a*) value in all extract-treated samples.It is conspicuous that meat color strongly influences consumers’ choice to purchase, since it is a reference of freshness and quality . The colThe obtained results are in harmony with those obtained by Kim et al. , who repThe sensory scores of spent hen meat patties treated with kiwi (5 and 7%), pineapple (5 and 7%), and a mixture of kiwi (5%) and pineapple (5%) are shown in Results of sensory examination revealed that there is a significant improvement in the case of all sensory attributes  of all extract-treated samples, as compared to their counterpart control samples. Among all extract-treated specimens, treatment of spent hen patties with a mixture of kiwi (5%) and pineapple (5%) achieved the higher sensory scores, however, lower consumer acceptability was recorded in samples treated with pineapple (7%), owing to over tenderization and the mushy texture of the product.Our results are in agreement with those reported by Kang et al.  and KadıLight micrographs images of the control-untreated spent hen meat patties, stained with H&E showed straight, nearly intact, closely bound, nucleated muscle fibers  displayed more degenerative changes in the muscle fibers  is important to obtain information about the modifications in the external morphology, texture, or orientation of muscle fibers . ScanninThese observations were in harmony with those reported by Ketnawa and Rawdkuen , who demLikewise, the topographical composition of chicken meat treated with lemon juice revealed irregular, broken, and separated muscle fibers . In addiMoreover, Rumondor et al.  reportedThe electrophoretic pattern of spent hen meat patties treated with kiwi (5 and 7%), pineapple (5 and 7%), and a mixture of kiwi (5%) and pineapple (5%) showed a reduction in the number and the intensity of protein bands that revealed an increase in the muscle proteolysis, when compared with control-untreated spent hen meat patties . Such anSimilar to the obtained results, Abdelrahman et al.  reportedIn another study, Liu et al.  reportedFrom the current investigation, it was observed that the treatment of spent hen meat patties with kiwi and pineapple extracts induced a significant reduction in the collagen content and shear force value, a significant increase in collagen solubility % with significant improvements to all sensory attributes, when compared with the control samples. The histological and topographical structure of all extract-treated spent hen meat patties displayed cracks and fragmentations in the myofibers, with clear degenerative changes in connective tissue. Additionally, the electrophoretic pattern of all extract-treated spent hen meat patties showed a decrease in the number and the intensity of protein bands, with an increase in muscle proteolysis. Accordingly, kiwi and pineapple extracts can be utilized by processors to increase the consumer acceptance of spent hen meat, in addition to the increase of its suitability, as a raw material, for the production of various chicken meat products.


Lophostoma currently includes eight species whose phylogenetic relationships have not been resolved. Integrative analyzes including morphological, molecular and chromosomal data are powerful tools to investigate the phylogenetics of organisms, particularly if obtained by chromosomal painting. In the present work we performed comparative genomic mapping of three species of Lophostoma , by chromosome painting using whole chromosome probes from Phyllostomus hastatus and Carollia brevicauda; this included mapping interstitial telomeric sites. The karyotype of L. schulzi (LSC) is a new cytotype. The species L. brasiliense and L. carrikeri showed interstitial telomeric sequences that probably resulted from expansions of repetitive sequences near pericentromeric regions. The addition of chromosomal painting data from other species of Phyllostominae allowed phylogeny construction by maximum parsimony, and the determination that the genera of this subfamily are monophyletic, and that the genus Lophostoma is paraphyletic. Additionally, a review of the taxonomic status of LSC is suggested to determine if this species should be reclassified as part of the genus Tonatia.The subfamily Phyllostominae  comprises 10 genera of Microchiroptera bats from the Neotropics. The taxonomy of this group is controversial due to incongruities in the phylogenetic relationships evident from different datasets. The genus  The phylogenetic relationships between the genera of this subfamily and even their positioning in relation to other subfamilies of Phyllostomidae are controversial, as different data sets show different phylogenetic patterns, which result in different taxonomic classifications [for review:7].The subfamily Phyllostominae  is composed of 10 genera with 25 speciesLophostoma d’Orbigny, 1836 includes eight species: L. silvicola d’Orbigny, 1836; L. brasiliense Peters, 1867; L. carrikeri Allen 1910; L. evotis Davis & Carter 1978; L. occidentalis Davis & Carter 1978; L. schulzi Genoways & Williams 1980; L. yasuni Fonseca & Pinto, 2004 and L. kalkoae Velazco & Gardner 20128, which range from southern Mexico to central Paraguay10.The genus 13. Chromosomal painting data have been successfully used in the reconstruction of karyotypic evolution among several groups of bats, including Phyllostomidae, which resulted in the correct identification of chromosomal homologies, the reconstruction of the phylogeny of the family and interpretation of the ancestral karyotype22.Morphological, molecular and chromosomal data, analyzed under an integrative taxonomy approach, constitute powerful tools for understanding the phylogenetic relationships between groups of organisms. Comparative analysis by chromosomal painting between species, associated with chromosome banding, can elucidate the types of intraspecific and interspecific rearrangements involved in the process of chromosomal differentiation that occurred throughout the evolution of taxaLophostoma show variable rates of karyotypic evolution where some species have highly conserved karyotypes such as L. silvicola 24 and L occidentalis 25 On the other hand, the genus also has species with highly rearranged karyotypes: L. brasiliense 29, L. carrikeri 30 and L. schulzi 31. Analyzing the species L. schulzi, Baker et al.29 found a karyotype so derived that none of the chromosomal arms proposed as primitive for the family were identified, suggesting that non-Robertsonian rearrangement processes would be involved in the differentiation of these karyotypes. In addition, most of the karyotypes were presented only in Giemsa conventional staining, preventing comparisons that could provide information about the types of chromosomal rearrangements that distinguish them.Species of the genus Lophostoma  were analyzed by chromosome painting using whole chromosome probes from Phyllostomus hastatus and Carollia brevicauda17. Chromosomal painting data for L. occidentalis16, L. silvicola20, Phyllostomus hastatus17, Gardnerycteris crenulatum16, Tonatia bakeri16, Tonatia maresi20, and Macrotus californicus15, were added to the phylogenetic analysis, in order to observe the relationships between Lophostoma species and their position within the Phyllostomini tribe.In the present work, the karyotypes of three species of the genus Lophostoma schulzi shows 2n = 26 NF = 34, comprising metacentric (pairs 6 and 9), submetacentric (pair 1), subtelocentric (7 and 8) and 7 acrocentric pairs . The X chromosome presents acrocentric morphology  and Carollia brevicauda (CBR) probes delimited 24 and 39 homologous segments in the genome of Lophostoma schulzi respectively. The number of signals per chromosome pair ranged from one to five.ric pair , subteloric pair , subteloric pair , subteloric pair , subteloLophostoma brasiliense shows 2n = 30 NF = 56, and the autosomal complement consists of 9 pairs of metacentric chromosomes , three submetacentric pairs  and two subtelocentric pairs (pairs 9 and 10). The X chromosome is subtelocentric of medium size , and two large subtelocentric pairs (pairs 1 and 12). The X chromosome showed acrocentric morphology  was 0.88, the retention index (RI) = 0.8594 and the homoplasy index (HI) = 0.12. Bootstrap values ranged from 61 to 100.The chromosome paintings by Sotero-Caio et al.ogy Fig. . For theLophostoma brasiliense (2n = 30 NF = 56) and Lophostoma carrikeri (2n = 26 NF = 46) analyzed here are in agreement with the studies previously carried out23, in specimens collected in Peru and for specimens collected in Suriname32. We described a new cytotype for Lophostoma schulzi 2n = 26 (6 sm/4 st + 16 a) for individuals collected in Juruti, State of Pará, Brazil (present study) that differs from the L. schulzi cytotype 2n = 28 (4 sm/6 st + 18 a), for individuals collected in Suriname30. Comparative analysis between L. schulzi karyotypes from Suriname and Brazil showed that the difference is due to a centric fusion/fission rearrangement. Chromosomal variations for populations of this species had not yet been reported, although they have already been recorded for other phyllostomid36.The data on the karyotype of the specimens of 38. The acrocentric form of X has been reported only for some genera, such as Micronycteris and Mesophylla28, a form admitted as a homoplasic character in these species because they are not closely related. In Lophostoma, the acrocentric X chromosome is found only in L. carrikeri and L. schulzi , similarly suggesting a homoplasic character, since these species are phylogenetically far apart  were also reported48. Telomeric hybridization was observed here at the ends of all chromosomes of species of the genus Lophostoma. Additionally, ITS were observed in L. brasiliense  and L. silvicola (LSI)20 the 18S ribosomal DNA mapped into a block of heterochromatin. The only exception is L. occidentalis (LOC)16, where the 18S rDNA maps to chromosomes corresponding to pairs 5 and 19 of Macrotus californicus  to karyotypes with many rearrangements (LSC).Through chromosome painting, using PHA and CBR whole chromosome probes, it was possible to establish the homeologies between the karyotypes of the species analyzed here. Additionally, we added the data previously obtained with these same probes in 22, we show that the LOC and LSI karyotypes are very similar to the PHA karyotype, differing only by the fission of PHA13. In addition to presenting autapomorphies, the LBR and LCA karyotypes share the syntenic arrangement PHA13/1/6 (pair 1 in both species). These species and also LOC and LSI share with PHA the pairs PHA1, 2, 3, 4, 7, 12 and a metacentric X. Finally, the LSC karyotype is the most differentiated in relation to other species, as most chromosomes are acrocentric and show a greater number of rearrangements in relation to the ancestral and PHA karyotypes. These data confirm the observation by Baker et al.29, that LSC has a derived karyotype in which none of the chromosomal arms proposed as primitive for the family were identified by G-banding.Considering the PHA karyotype as a reference, as it is close to the ancestral karyotype proposed for Phyllostomidae20 mapped the Tonatia saurophila karyotype collected in the Brazilian Amazon with PHA and CBR whole chromosome probes. Sotero-Caio et al.16 mapped the same species, collected in Central America, with MCA whole chromosome probes. Recently the specimens of T. saurophila were reclassified49, considering only specimens from Jamaica as belonging to this species, while specimens located in Central America and northeast of South America were considered to belong to the species Tonatia bakeri (TBA), while specimens from the Amazon would belong to the species Tonatia maresi (TMA), with the Andes being the barrier that separates these new species. The presence of karyotypic differences could be an additional diagnostic element to define these species. Thus, we converted the probe mapping data from MCA to PHA in TBA by Sotero-Caio et al.16 using Fig. 20. Sotero-Caio et al.16 had already noticed the difference in morphology of the TBA4 pair  between the karyotypes, suggesting a pericentric inversion. After comparing the mapping results of the two species, we found three chromosomal rearrangements between these karyotypes: (1) the difference between TBA4 and TMA6 is actually due to an insertion of a segment of the PHA10 chromosome (= MCA3) into the short arm of TBA4; (2) Insertion of a segment of the PHA7 chromosome (= MCA4) into the long arm of TBA3 (= TMA5); (3) Inversion in the long arm of TBA2 (= TMA4), where the syntenic group corresponding to PHA1  are joined in TMA and separated (MCA5 and MCA10) in TBA. Therefore, these differences reinforce the specific status of the Central American (TBA) and South American (TMA) taxa and were included in the data matrix . The Cytogenetics Laboratory from Centro de Estudos Avançados da Biodiversidade (UFPA) has permit number 19/2003 from the Ministry of Environment for sample transport and permit 52/2003 for using the samples for research. Sample collections were authorized by Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) and Secretaria de Estado de Meio Ambiente do Pará (SEMA-PA) under permit 020/2005 (Registration: 207419).All experimental protocols were approved by the Ethics Committee from Para Federal University  under Permit 68/2015. All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines  at 60 °C and staining with Wright’s solution57. C-banding was performed according to literature58. Karyotypes were arranged in decreasing order of chromosome size.Chromosomal preparations were obtained by the direct bone marrow extraction method59. The probes were labeled with dUTP-biotin labelled antibody by nick translation and later detected with avidin-Cy3 or FITC labelled antibodies.FISH using telomeric probe labeled with digoxigenin (All Human Telomere—Oncor), and detected with FITC-Cy3 labelled antibody, was performed according to the protocol provided by the manufacturer. The 18S rDNA probes were amplified by PCR using the probes NXS1 and NS8, as described in literatureCarollia brevicauda and Phyllostomus hastatus17 were amplified and labeled by DOP-PCR61 and hybridized following previously described61. Briefly, slides were incubated with pepsin enzyme solution, washed in 2× SSC solution and serially dehydrated with ethanol . The slides were then aged in an oven at 65 °C for two hours, denatured in formamide 70%/2× SSC for 50 s and mounted with hybridization solution  for three days. After hybridization and stringency washing, biotin-labeled probes were detected with avidin-Cy3 or avidin-FITC . The slides were mounted with Vectashield antifading solution (Vector Lab) and DAPI  and the images were captured with the aid of the Zeiss Axiocam CCD camera controlled by the Axiovision 3.0 software, coupled to a Zeiss Axioplan 2 microscope. Chromosomes were identified according to their morphology and banding patterns interpreted from DAPI  staining images edited in grayscale format.Whole chromosome probes from 62. All characters were weighted with the same weight, based on the equal probability of occurrence of chromosomal rearrangements. We searched the most parsimonious phylogenetic tree, which was obtained using the exhaustive search. The robustness of each node was evaluated by bootstrap estimation of 1000 repetitions. Macrotus californicus15 was used as outgroup and as ingroup the three species studied here and L. occidentalis16, L. silvicola20, Phyllostomus hastatus17, Gardnerycteris crenulatum16, Tonatia maresi20, and Tonatia bakeri16.We used syntenic segments and shared chromosomal associations to establish a matrix of characters (Supplementary Table Supplementary Table 1.


In reconstructive surgery using artificial materials after wide resection, soft tissues are usually adjacent to metal surfaces or mesh. The purpose of this study was to provide histological evaluation of the soft tissues adjacent to the metal surfaces of megaprostheses and mesh. Tissues from revision surgery of megaprosthesis and from wide resection after recurrent thoracic wall sarcoma were used. Histological analysis was evaluated by hematoxylin/eosin (HE) and Masson’s trichrome staining, and by immunohistochemical staining for markers including cluster of differentiation 68 (CD68), vimentin, collagen type and S100A4. Soft tissue adherence to the smooth metal surface of Ti alloy was not observed. On the surface of capsule, CD68- and vimentin-positive cells formed a thin layer. In contrast, soft tissue adherence to a rough-surface cobalt chrome alloy was observed. Capsule was not apparent for this tissue, in which CD68- and vimentin-positive cells were aggregated randomly. In the resected tissues of recurrent chest wall sarcoma, muscles showed connections to connective soft tissues but did not invade to the inside of the mesh. Around the polypropylene mesh, large numbers of CD68- and vimentin-positive cells were seen. On the ePTFE, small numbers of CD68-positive cells were observed, while a larger number of the cells were vimentin positive. High accumulation of S100A4-positive cells was observed at the metal surface and polypropylene surface. Cells were strongly positive for CD68 and vimentin in tissues adjacent to metal and mesh surfaces. Macrophages and vimentin may play important roles in the foreign body reaction to metal and mesh, and so may contribute to encapsulation and fibrosis. Bone and soft tissue sarcoma resection with a wide margin constitute some of the most successful cancer treatments supported by reliable evidence ,2. Wide Endoprosthesis is a common device for reconstruction after bone resection for bone tumors . This stTo recover function, anatomical reattachment of detached bone, muscles, or tendons to metal surfaces is the ideal process and should be considered. Depending on the available residual bone or loss of soft tissues, metal surfaces are connected by bone, tendon, muscle, or artificial materials. Reconstruction of knee extension is the most important theme in megaprosthetic replacement of the proximal tibia. A variety of reconstruction methods using artificial ligament, mesh, composite allograft, etc., have been reported . In all TM Mesh), polyester , or similar materials, often is used for reconstructive surgery. Mesh is readily moldable and commonly is used for chest wall and abdominal wall reconstruction to maintain the anatomical cavity despite the occurrence of wall defects following wide resection. To inhibit organ adhesion, coating of the mesh (on the organ side) with expanded polytetrafluorethylene (ePTFE) has been used; one such composite material is BardTM ComposixTM Mesh. Moreover, mesh sometimes is applied for reconstruction after wide resection of bone and soft tissue malignant tumors in extremities. In one such strategy, polypropylene mesh is wrapped around the prosthesis, and the cut ends of muscles are sewn onto the mesh to create muscle insertions [Mesh, which typically is constructed using polypropylene  has not been the focus of previous studies and has been reported only rarely. Improvements of soft tissue integration to artificial materials such as metal, mesh, or novel materials would require further characterization, for which histological analysis would be the first step. Therefore, the purpose of this study was to provide histological evaluation of the soft tissues adjacent to metal surfaces and polypropylene mesh with ePTFE, employing tissues from revision surgery using megaprostheses following wide resection after recurrent thoracic wall sarcoma.This study was approved by the Ethics Committee of the Mie University Graduate School of Medicine . All procedures performed in studies involving human participants were in accordance with the ethical standards of the Ethics Committee of Mie University and with the Helsinki declaration of 1975. Written, informed consent was obtained from each patient. For patients below the age 19 y, informed consent was obtained from their parents or legal guardian.A 13-y-old woman received right-knee megaprosthesis replacement  after extraarticular wide resection due to osteosarcoma of the distal femur . After 6A 38-y-old man received knee megaprosthesis replacement  due to recurrence of Ewing sarcoma of the proximal femur. After 10 y, the patient underwent a revision surgery in response to infection at the HMRS. After another 10 y, the stem of the HMRS broke, necessitating further revision . After tTM ComposixTM Mesh was used for the chest wall reconstruction. One year later, a further recurrence was observed, necessitating another wide resection  of the chest wall. When the MPNST recurred 3 y after first wide resection, Bardesection . These rThe tissues were fixed with 10% neutral buffered formalin and embedded in paraffin according to conventional methods, and then sectioned as 4-µm thicknesses using a microtome.The sections were stained with Masson’s trichrome using Muto Pure Chemicals Co., Ltd.,  reagents as follows. The sections were deparaffinized and incubated with the first mordant for 30 min, followed by incubation in Mayer’s hematoxylin solution for 3 min. After washing in water, the sections were incubated with 0.75% Orange G solution for 1 min. The tissue sections then were submerged in 1% acetic acid and incubated with Masson’s stain solution B for 20 min. The sections again were submerged in 1% acetic acid, incubated with 2.5% phosphotungstic acid for 10 min, submerged yet again in 1% acetic acid solution, and incubated with aniline blue solution for 30 min. The sections then were washed in ethanol, and dipped in xylene.2O2 to eliminate endogenous peroxidase activity. After washing, the slides were incubated for 30 min at 24°C with an anti-mouse immunoglobulin that had been conjugated with horseradish peroxidase (HRP) using an immuno-enzyme polymer . All specimens were visualized in 3,3’-diaminobenzidine tetrahydrochloride (DAB) solution containing hydrogen peroxide. After washing in water, the sections were counterstained with hematoxylin.For immunohistochemistry (IHC), sections were subjected to paraffin removal, and antigen retrieval was conducted by heating the sections for 10 min in 10 mM citrate buffer (pH 6.0) at 120°C using a pressure cooker . The slides then were incubated overnight at room temperature with primary antibodies specific for vimentin , CD68 , CD3 , CD20 , or collagen type I , diluted 1:500; Catalog F56, Kyowa Pharma Chemical Co., Toyama, Japan). After washing, sections were incubated for 30 min at room temperature in methanol containing 0.3% HRevision surgery for the KMLS is shown in Revision surgery for the HMRS is shown in HE and Masson’s trichrome staining showed the presence of a rough tissue surface along the rough metal surface. This rough tissue surface (arrows) conformed to the metal processes; upon peeling away from the metal, the tissue shape slumped , arrow. TM ComposixTM Mesh without MPNST tumor are shown in Resected tissues with BardThe numbers of S100A4 (fibroblast-specific protein-1)-positive cells per high-power field (HPF) at the area adjacent to the metal surface, ePTFE, and polypropylene and their deep areas were counted. At the area adjacent to the KMLS, there was a uniform distribution of S100A4-positive cells, with an average of 200.3 cells/HPF , 1–3, anIn this study, we performed histological analysis of the soft tissues adjacent to megaprostheses and mesh using surgical tissues recovered from clinical cases. Biomaterial implantation induces foreign body response, and the degree of this response differs depending on the properties of the implanted material. The biological process of the foreign body response follows a defined course that includes  provisional matrix formation, acute inflammation, chronic inflammation, and fibrous capsule formation . MacrophTitanium and titanium alloys are thought to be bioinert for bone connection. Bone tissues have been shown to regenerate along titanium surfaces . To enhaIn Other studies have examined the mechanical strength of soft tissue integration onto metal surfaces in vivo. In 1982, Bobyn et al. measured peeling strength using implants in adult mongrel dogs . These rMesh frequently induces macrophage accumulation due to chronic foreign body reaction. The degree of this response differs based on the mesh material, number of filaments, and the presence or absence of knots ,47. OsanThe ideal material for prosthetic implants would exhibit rapid wound healing, minimal chronic inflammation, minimal foreign body reaction, resistance to infection, and rapid soft tissue integration ,47. FurtThis study employed histological analysis of tissues adjacent to megaprostheses and mesh in tissues recovered from clinical cases. Staining for CD68 and vimentin was strongly positive in the tissues adjacent to metal and mesh. We infer that macrophages and vimentin may play important roles in the foreign body reaction to metal and mesh, and that these reactions contribute to encapsulation and fibrosis. It is expected that surface modifications of metal and mesh surfaces will improve cell reaction and tensile strength of regenerated soft tissues adjacent to implanted protheses.The number of patients was small, and alloy was different. However, revision surgery of megaprosthesis and wide resection including mesh after recurrent thoracic wall sarcoma were extremely rare. We believed this is a chance to provide histological evaluation of the soft tissues reaction adjacent to metal surfaces and polypropylene mesh in human.