This research examined the performance of functionally graded two-stage fibrous concrete (FTSFC) against modified repeated falling-mass impacts. This study led to the concept of creating improved multiphysics model of fibre composites with better impact resistance for potential protective constructions. FTSFC was developed based on the bio-inspiring strength of turtle shells. The excellent impact resistance of FTSFC was accomplished by including a larger quantity of steel and polypropylene fibres in the outer layers. At the same time, one- and two-layered concrete were cast and compared to evaluate the efficiency of three-layered FTSFC. To minimize the dispersed test results, a modified form of the 544 drop-mass impact test was recommended by the American Concrete Institute (ACI). The modification was a knife-edge notched specimen instead of a solid cylindrical specimen without a notch. This modification predefined a crack path and reduced the dispersion of results. Cracking and failure impact numbers, ductility index, and failure mode were the testing criteria. The suggested modification to the ACI impact test decreased the coefficient of variance, showing that the dispersion of test results was reduced significantly. This study led to the concept of creating improved, fibre composites with better impact resistance for potential protective constructions. The construction industry is developing rapidly due to novel composite materials and methods. One of the most emergent methods is the bionic-inspired design of functionally graded composites. These biomaterials have recently earned attention from researchers owing to their novel hierarchical structures and high impact strength . ExploriConstruction materials such as concrete are the most commonly used in the building sector due to the growing civilian and military infrastructure needs. A drastic increase in living standards, combined with a sharp rise in the global population, can be attributed to this development . The curFibre-reinforced concrete (FRC) is a frequently used composite material in the construction sector, since it increases the efficiency of concrete buildings subjected to various impact loadings. FRC is a promising material for modernizing various impact-resistant applications in critical structures, vehicle crashes on transportation structures, flooring, a long-haul viaduct, military facilities, radioactive storage, and airstrip pavements. The most vulnerable buildings are expected to be unaffected by a single powerful explosion, but structures subjected to repeated collisions exhibit damage, resulting in collapse. As a result of increasing artificial hazards, there has been a rising need to enhance the structural integrity of infrastructure exposed to repeated impacts and explosions. On this basis, several researchers have been employed to improve the FRC materials, which can exhibit a superior impact resistance to conventional concrete. The development of material technology and unification has enhanced the emergence of a new named composite: two-stage fibrous concrete (TSFC) .TSFC is a unique type of concrete that is manufactured using a different casting technique than traditional concrete. Many distinct names throughout the world are used for two-stage concrete: Colcrete, Polcrete, Arbeton, Naturbeton, prepacked concrete, grouted aggregate concrete, and injected aggregate concrete ,13,14,15The advancement of material technology and their unification has resulted in novel composites known as FGC. Fibrous FGC is a novel concrete composite with enhanced mechanical characteristics that ensures the required behavior by altering its properties. Greater density with a stronger fibre matrix is possible in functionally graded fibrous concrete (FGFC), which has outstanding toughness, uniaxial tensile strength, and impact resistance ,29. MoghSeveral test techniques have been described in the literature  for evalSeveral researchers have recently utilized the ACI 544 repetitive falling drop weight or mass impact (RFDWI) test to assess the impact resistance of fibrous cementitious materials ,51,52,53A single-point impact on the specimen increased the chance of inaccurate findings—the point of impact could be a soft cement matrix or a hard coarse aggregate;Cracks were permitted in any direction and anywhere on the specimens—this strengthened the test subjectivity when cracking was inspected visually;Due to the lack of a standard, the test findings were dispersed;The specimen base, as well as the apparatus lug, were characterized as failures, and even with excessive fracture width, this failure observation could produce a recurrent impact on the specimen;Preparation of specimens was not based on any standard, so the specimens may have had mold-faced surfaces.Despite the numerous benefits of the ACI drop-weight impact test, the dispersed results were the primary disadvantage that must be addressed.Consequently, this test made it simple to determine the relative energy engrossment of various materials. The pilot investigations of the drop-weight impact testing on different kinds of concrete are shown in It permitted the formation of cracks in any direction, complicating the identification of the initial visual crack;Since concrete has a heterogeneous material feature, the centre point that was immediately exposed to the focused force could be a coarse solid aggregate grain or a soft mortar region. As a consequence, results that will not accurately reflect the material’s impact strength could be achieved.In this study, the leading two causes of inaccuracy were examined, and changes were proposed accordingly. Badr and Asour et al.  identifiThere is a shortage of adequate information in the bibliography regarding modified testing procedures for the ACI drop-weight test. Despite this, researchers worldwide have utilized different statistical methods to evaluate the substantial dispersion in ACI drop-weight test data. It is fascinating to perform the modified impact test and reach a logical conclusion to minimize dispersion outcomes. However, very little study has been conducted to decrease dispersion outcomes by adding granular bedding, and gaps in this research field are still open. To fill these research gaps, this study sought to evaluate the impact performance of FTSFC by performing a modified impact test. A simple technique was suggested to minimize dispersion effects by utilizing a notched specimen and a cross knife plate instead of a non-notched specimen and a steel ball. The impact number related to cracking and failure, failure mechanism, and ductility index were studied in detail in our research.2/kg; the consistency was 30.8%; and the initial and final setting times were 32 and 550 min, respectively.The cement used in this research had a specific gravity of 3.14 as per the IS 1489-2015 standard . The conNatural river sand was used as the fine aggregate, meeting the requirement of IS 383-2016 ; the gra3, the specific gravity was 2.6, and the water absorption percentage was 0.56.The coarser aggregate utilized were natural gravel with a size of 12.5 mm meets the requirement according to IS 383-2016 . The appThe commercial superplasticizer Tech Mix 640 was utilised to reduce water and extend grout time in the plastic stage. A grout fluidifier typically is composed of a water-reducing additive at a suggested dose of 1% by cement content . The watFibre is widely used as a component in concrete reinforcement due to the many advantages it provides in this application. A new geometrically shaped macro polypropylene fibre (PF) and steel fibre (SF) were utilized in this study; the unique PF was 45 mm in length and 0.8 mm in diameter, with a tensile strength of 500 MPa; and the hybrid hooked-end crimped SF had a length of 50 mm, a diameter of 1.0 mm, and a tensile strength of 1200 MPa. The appearances of the PF and SF used in this research are illustrated in Twelve different concrete mixes were developed in the present study to assess their impact resistances. A series of grout blends were developed to select the optimal grout based on the cone-test efflux time, which met the compression strength and flowing grouting criteria. The optimized grout efflux time ranged from 35 to 40 s, as per the American Society for Testing and Materials (ASTM) C939 standard . The optA total of 180 cylindrical specimens were produced, with 15 per mixture. To assess the impact strength, cylindrical specimens with a diameter of 152 mm and a height of 64 mm were utilized. The FTSFC step-by-step casting technique involved three essential steps, as shown in In accordance with ACI Committee 544–2R, a modified drop-mass impact test was performed to assess the impact strength of the FTSFC specimens . The tesFor each type of combination, three cubical specimens were cast and tested for compressive strength in accordance with the standards of IS 516 . The comCompared to PAC, three-layered FTSFC reinforced with various fibre doses showed an improvement in compressive strength that varied from 5% to 54%. The lowest compressive strength found in this category was for the T-FG6 mix, which had a compressive strength that was 5.2% greater than the PAC mix, as shown in This study investigated the impact behavior of FTSFC specimens under falling mass impact. The impact numbers causing the initial crack (Q1) and the final crack (Q2) are listed in The effects of a single-layered specimen on the impact strength are shown in The effect of fibre addition and its distribution in concrete was the most common cause of a rise in Q1 and Q2. Uniformly dispersed fibres inhibited micro/macrofracture development, allowing for improved stress homogeneity in the concrete by distributing stress concentration over a larger region. The fibre bridging effect was accounted for by changing the crack direction, intensifying the crack progression, and minimizing the crack width . Cracks The crack propagation in the D-PF-SF specimen was slow when compared to the D-SF-PF specimen. The D-PF-SF specimen contained a 2.4% dosage of PF in the top layer and SF in the bottom layer. The fracture began on the top surface and rapidly expanded into the second layer under falling-mass impact. The second layer resisted the crack propagation more effectively due to the presence of SF. Fibre crack-bridging activity of SF, which improved the Q1 and Q2 of the D-PF-SF and D-SF-PF specimens, substantially impacted the mechanism of crack stabilization. This process was essential in enhancing impact strength by improving debonding, sliding, and drawing out of the fibre and delaying crack formation. Consequently, the impact strength of two-layered FTSFC specimens was lower than single-layered S-SF specimens. This could be due to more SF being distributed in the entire cross-section in the one-layered concrete specimen, while the two-layered FTSFC specimen contained SF in only one layer.The Q1 and Q2 values for the three-layered FTSFC specimens comprising mono- and hybrid SF and PF in different combinations are shown in The recorded Q1 and Q2 values for the T-FG1 specimen were 101 and 357, respectively. When compared to the PAC specimen, the observed values improved by 5.61 and 11.16 times, respectively.For the T-FG2 specimen, the recorded values for Q1 and Q2 were 106 and 501, respectively. The observed values were increased by 5.88 and 15.65 times, respectively, compared to the PAC specimen.The recorded Q1 and Q2 values for the T-FG3 specimen were 94 and 279, respectively, and these values were increased by 5.22 and 8.71 times, respectively.The Q1 and Q2 values for the T-FG4 specimen were 112 and 608, respectively. These values tended to increase by about 6.22 and 19 times, respectively.The recorded Q1 and Q2 values for the T-FG5 specimen under the optimal circumstances were 116 and 742, respectively. The recorded values increased by 6.44 and 23.18 times, respectively.The Q1 and Q2 values for the T-FG6 specimen were 95 and 307, respectively. The recorded values were increased by 5.27 and 9.59 times, respectively.For the T-FG7 specimen, values of 105 and 389 were recorded for Q1 and Q2, respectively. The recorded values were increased by 5.83 and 12.15 times, respectively.All FTSFC specimens exhibited an excellent impact resistance compared to the PAC specimens. The highest Q1 and Q2 values were recorded for the T-FG5 specimen. This was due to the higher dosage of SF provided in the top and bottom layers, which increased the tensile strength of these layers. As a result of the compressive waves reflected produced by the falling mass, these layers could resist a more significant number of tensile waves formed at the specimen’s top surface. The ductile FTSFC specimen was designed to withstand massive tensile-stress waves mainly in the upper and bottom layers while also decreasing the energy wave via microcracking. Using a higher dosage of SF and PF in the top and bottom layers led to enhanced tensile strength and provided an additional impact-energy absorption mechanism owing to membrane action, which distributed impact stress over a larger area . The perThe IDI was defined by the ratio of Q2 to Q1, and IDI values for all mixtures are shown in The failure of the FTSFC at the top and side face under impact loading is shown in The orientation of the fibres can affect the impact resistance of the composite ,84,85. AContact damage, failure of the matrix, failure of fibres, and additional fibres resulting from damage were seen in the specimens due to loading/delamination. These damaging effects occurred quickly, and it was thus difficult to explain their correct order. The COV analysis showed the distribution of the impact-test findings. Increased and decreased COV values resulted in increased and decreased dispersion of impact-test findings, respectively. In general, a smaller COV was preferable in all cases because it reflected a more precise assessment. Similarly, modified impact-testing results revealed a reduced COV for all 12 mixes, ranging from 13.8 to 26.5% for Q1 and 7.1 to 22.5% for Q2. As shown in The S-SF specimen demonstrated the greatest compressive strength, with a 59.6% improvement over the reference specimen (PAC). The T-FG2 specimen, which had 2.8% SF in the top and bottom layers and 1.6% in the intermediate layer, had the second-greatest compressive strength increase at 54.4%. As a result, single-layered concrete outperformed three-layered FTSFC in compression tests. On the other hand, steel fibres contributed more to improving strength than PF, independent of the fibre scheme or the number of layers.The reported Q1 increased by approximately 5.8 and 3.8 times for the S-SF and S-PF specimens, respectively. In comparison to PAC, the reported Q2 increased by about 17.5 and 5.2 times. For both Q1 and Q2, however, the effect of SF was greater than that of PF. This was caused by the introduction of fibres, which improved the matrix’s tensile capacity by providing high tensile-stress absorbance across fractures through crack spanning.The T-FG group of specimens had higher Q1 and Q2 records than the PAC sample, which was expected due to the fibre-matrix reinforcing effect. The T-FG5 combination from this group had the most significant increases in Q1 and Q2, by about 6.4 and 23.2 times, respectively. This was due to the greater SF dose in the top and bottom layers, which were subjected to higher impact stresses due to the immediate contact with the supplied drop weight and the supportive base plate. Furthermore, the crimped and hooked-end structure of SF and its considerably greater tensile strength than PF contributed to its enhanced bond strength. The second-highest Q1 and Q2 values for three-layered FTSFC, recorded for the T-FG4 specimen, were 112 and 608, respectively. These values tended to increase by about 6.22 and 19 times, respectively. The third-highest recorded values were for the T-FG2 specimen, which had a Q1 and Q2 of 106 and 501, respectively. The observed values were increased by 5.88 and 15.65 times, respectively, compared to the PAC specimen.The PAC had a ductility index value of 1.8, which indicated brittle failure. The ductility index value of all fibre specimens varied from 2.4 to 6.4, indicating a higher postcrack resistance. Moreover, a higher ductility was achieved by increasing fibre content in the top and bottom layers while reducing it in the intermediate layer.Better controlled cracking behaviour was achieved by using notched specimens and load-transmitting plates. The cracks in notched specimens mainly started and spread along the edges of the notches, while the specimens examined per the ACI 544-2R method had numerous randomly dispersed cracks. This type of controlled activity would make it simpler to identify criteria for accepting or rejecting the findings of the specimens tested based upon their cracking pattern, reducing the dispersion of the results even further.In comparison to the ACI technique, the modified-impact findings were considerably more significant. For Q1, the percentage difference between the ACI and modified impact test findings varied from −6 to 100%, while for Q2, the percentage difference ranged from 11.8 to 100.4%. Compared to the ACI test technique, the estimated COV values for all 12 mixes were reduced by 27.4 to 64.21% for Q1 and 6.3 to 70.16% for Q2. Consequently, the suggested impact-test modification can enhance the reliability of findings, is simple to perform, and contributes to emerging material technology.This study used a modified impact test for functionally graded two-stage fibrous concrete (FTSFC) against repeated low-velocity impacts. To minimise the dispersion of experimental results, a cross notch was created on the specimen, and a knife-edge steel bar was horizontally positioned to provide a parallel-line impact force rather than the usual single-point impact. Various mono- and hybrid schemes were derived from testing the impact resistance of single-, double-, and triple-layered concretes made of steel and polypropylene fibres. According to the thorough investigation, the following were the most important outcomes:


Shape memory polymer (SMP) products have been developed for application as crack closure devices in concrete. They have been made from PET in the form of both fibres and hollow tubes. Here, manufacturing methods using die-drawing and mandrel-drawing to induce shape memory are reported. The fibre-based devices are incorporated into concrete and, upon triggering, exert shrinkage restraint forces that close cracks in the concrete. The evolution of shrinkage restraint force in the fibres as manufactured was measured as a function of temperature, showing stresses in excess of 35 MPa. Tendons consisting of fibre bundles are incorporated into concreate beams subjected to controlled cracking. When activated, the tendons reduce the crack widths by 80%. The same fibres are used to produce another class of device known as knotted fibres, which have knotted ends that act as anchor points when they incorporated directly into concrete. Upon activation within the cracked concrete, these devices are shown to completely close cracks. The tubes are used to enclose and restrain prestressed Kevlar fibres. When the tubes are triggered, they shrink and release the prestress force in the Kevlar, which is transferred to the surrounding concrete in the form of a compressive force, thereby closing cracks. The Kevlar fibres also provide substantial reinforcement after activation. The devices are shown to be able to partially and fully close cracks that have been opened to 0.3 mm and achieve post-activation flexural strengths comparable to those of conventional reinforced and prestressed structural elements. Finally, a preliminary life cycle assessment study was used to assess the carbon footprint a nominal unit of concrete made with SMPs fibres compared to conventional concrete. Concrete is one of the most used materials in the built environment, with an annual production of 4.1 Gt in 2019 .Being a relatively brittle material and being very weak in tension, cracks are to be expected when significant tensile stresses are induced. A concrete structural element may be subjected to a combination of stresses which could cause the formation of cracks where and when the tensile strength of the material is exceeded. Cracking in concrete can also be caused by early age thermal effects, structural loading, differential creep and shrinkage, chemical changes within a structural element, and environmental conditions such as earthquakes or floods. Whatever the cause, surface and internal cracks are inevitable during the service life of a concrete structure .Cracks undermine structural integrity and can lead to reduced durability by allowing the ingress of water and other substances . For exaAlthough an increased effort has been made to design more durable structures with longer service lives, it remains the case that many modern concrete structures require significant maintenance within the first ten to fifteen years of their service lives . Such reThe above considerations form a strong case for the introduction of technologies that combat cracking in concrete, either by preventing the formation of cracks or stopping their growth.One way of minimising or controlling the width of cracks in concrete structural elements is by the addition of shrinkage reducing agents and supplementary cementitious materials, such as MgO, fly ash, crystalline admixtures ,11,12, oPolymers have played an important role in civil engineering. For example, they are used in fibres, pipes, geotextile materials, and cladding products .Recycled polymer (PET) particles and fibres have also been used in concrete mixes. These have been used to partially replace conventional aggregate and to enhance the resilience of the material ,19,20. FThe incorporation of PET into asphalt pavements has also been considered. In Lugeiyamu et al. it was shown that the replacement of 10% of asphalt binder in stone mastic asphalt mixture enhances the resistance against moisture damage and better performance with regards to low and medium temperatures and fatigue loading .In general, the incorporation of recycled PET into particulate composites makes the material more sustainable, gives economic benefits and often enhances engineering properties ,25,26.In this paper, we report a series studies undertaken by the authors to develop crack closure systems for concrete structural elements that use components formed from shape memory polymers (SMPs). Previous publications on this work in civil-engineering journals have concentrated on the structural properties of the concrete elements see . By contThe stretching and alignment of macromolecules is the physical basis for the operation of SMPs, and also a means of producing polymer with increased stiffness and strength. The latter factor drove the development of polymer product with aligned molecules—the so-called oriented polymers—that began in the 1970s. Spinning processes have been used for many years to produce oriented polymer fibres of high strength and stiffness ,28. For Die-drawing was originally developed using conical converging dies ,32 to prThe above solid-phase processes all have potential for the manufacture of SMPs. Die-drawing is more attractive than hydrostatic extrusion as it can be adapted as a continuous process.The selection of the polymer to be used in this application is governed by several factors. To be practical, the polymer must be commercially available on a mass scale so as to be economically feasible. It needs to have sufficient strength and stiffness. If it is amorphous, this implies that it must be below its glass transition temperature under service conditions. It must also function as a shape memory polymer. The mechanism for this effect is the recovery of large strain in its amorphous regions, which can be inhibited in semi-crystalline polymer. These considerations lead us to a low crystallinity polymer with a glass transition above room temperature. When used as an SMP, this form of polymer operates by being deformed above its glass transition temperature and the cooled to prevent recovery; recovery then occurs when the polymer is reheated to its ’trigger’ temperature, which is near or at the glass transition. This introduces another limitation on the choice of polymer, as its trigger temperature must be compatible to its use in setting concrete. Practically this limits the glass transition temperature to lower than the boiling point of water. There are few mass-market amorphous polymers with glass transition between 20 and 100 °C. Some grades of polymethylmethacrylate (PMMA) have glass transition in this range, but preliminary experiments gave poor performance as an SMP. PET has a glass transition temperature of around 90 °C and has the advantage of being recyclable, opening up the potential for the use of recycled polymer in these applications. We therefore made this choice of polymer.We have adopted the die-drawing process to produce oriented fibres from a commercial PET (Dow Lighter C93) obtained as granules. The process was in two stages.First, isotropic fibres were made by melt extrusion. The extruder was a Killion S1748, with pressure and screw speed set, respectively, at 30 bar and 15 rpm. Temperatures varied from a maximum of 280 °C to 270 °C at the die head. The material exited through a 4 mm internal dimeter circular die at a haul-off speed of 5 m/min. It was then cooled in a glycerol bath at room temperature. The final product was of diameter 1.8 mm.−1 in the die) The oven and die temperatures were then both decreased from 80 °C in 1 °C increments until failure or stress whitening of the fibre were observed. This occurred at 75 °C. The oven was then held at this temperature while the die temperature was incremented until an unvoided fibre was produced. The temperatures established using this procedure were 75 °C in the oven and 80 °C in the die. Once the fibre had left the die, it drew further as it cooled in air between the die and the caterpillar. Its final diameter was 0.9 mm, corresponding to an extension ratio of 4.0. Haul-off force was monitored throughout the process and was found to be 80 N at its start, reducing to a value of 50 N as the process became stable These loads correspond to stresses of 99 and 62 MPa, respectively, and are beyond the yield strength of 55 MPa stipulated in manufacturer’s data sheet.Next, the material was die-drawn. The isotropic fibre, having cooled to room temperature, was heated by being pulled through a fan-assisted oven of length 1.5 m. The next stage was the die-drawing, as the fibre enters a converging conical die with cone semi-angle 30° and exit diameter 1 mm, maintained at a controlled temperature see . The proThe performance of these fibres in terms of shrinkage restraint force has been assessed using an apparatus consisting of a small chamber containing a single fibre, held outside the chamber by grips, one of which incorporates a load cell. The chamber was heated by compressed air at a controlled temperature and both the force and temperature were monitored see . TemperaFor SMP devices to be discussed below, PET tubes were manufactured using the same commercial grade of PET and the same Killion extruder as was used for the fibres above. First, tubes were extruded. Operating conditions were set at a screw speed of 70 rpm, pressure of 200 bar, maximum extruder temperature 280 °C, die head temperature of 260 °C and haul-off speed 500 mm/min. The melt was extruded through a circular section die of diameter 14 mm, with an internal pin of diameter 4 mm to create a central circular hole. The final dimensions were outer diameter 13 mm and inner diameter 6 mm.The tubes were then die-drawn to obtain oriented product. After heating in an oven, 1 m lengths of extruded tubes were drawn through a pair of dies. As shown in The SMPs described above have been used to create devices that have been used in concrete for civil engineering applications. This has been the result of collaboration between civil engineers and polymer engineers. The performance and effectiveness of the devices have been reported in mainly civil engineering journals where only summary details of the polymer technology were given. The purpose of the present paper is to discuss and report the polymer aspects of the work. However, we consider it necessary to give some summary details of the devices’ effectiveness. This represents the purpose of this section, within which the civil engineering publications are referenced.× 25 × 25 mm) with an axial void to accommodate the tendon. Each specimen was cast and cured, and then the PET tendon was installed in the void and clamped at the beam’s extremities. Each beam was loaded in flexure to create a crack of known crack mouth opening displacement (CMOD) and then demounted and placed in an oven to release the shape memory potential. It was shown that the activated tendons were able to close serviceability sized cracks in these small plain mortar beams, and that the system provided favourable conditions for autogenic healing [A crack closure system based on SMP tendons was proposed by Jefferson et al. (2010), and further developed by Dunn et al. (2011) ,40. In t healing . This eaIn order to scale-up the system and prove its viability for structural elements, larger scale tendons were needed. These tendons were formed from PET fibres, manufactured as described in Two beam configurations were used, namely one with PET tendons alone and the other with accompanying steel reinforcement. During testing, the surface crack opening displacements were measured using optical microscopy, and a DIC system was used to measure strains on one side of the beam. In addition, a number of plain concrete ‘control’ beams, without tendons or reinforcement, were cast and tested. A typical load-CMOD response of a beam with a tendon (denoted PET) is compared with that of a control specimen in Figure 6. This illustrates the sequence of responses exhibited by the PET specimen during initial loading (1–2), unloading (2–3), activation (3–4), reloading (4–5) and unloading (5–6). It may be seen that CMOD reduced substantially when the tendon was activated, and that this was accompanied by a significant regain of the flexural stiffness. and strength.PET tendons were used both as the sole means of reinforcement and in conjunction with steel; configurations are shown in Whilst the multi-fibre tendon assemblies proved able to close cracks in plain concrete specimens and provide some post-activation flexural capacity, the strength was of similar magnitude to the flexural capacity of the plain concrete beam but with substantially lower stiffness. The tests with the combined PET and steel reinforcement showed that the delayed prestress was insufficient to bring about full crack closure. Therefore, research was undertaken to develop a hybrid tendon system that could not only close cracks but also provide substantial levels of post-activation reinforcement and prestressing. The solution developed was the hybrid tendon described in the following subsection.A hybrid tendon comprises a pretensioned Kevlar core and a restraining SMP tubular sleeve. The die (or mandrel) drawing technique used to form the latter was described in The process used to manufacture the hybrid tendons shown in  is descrThe ability of the system to close cracks is illustrated in Additional research considered PET knotted shape memory polymers (PET k-SMP) in the form of filaments manufactured as described above in When casting the specimens (using prismatic moulds) for crack closure, it was decided that two distributions of sample would be made—those with an aligned distribution (AD) where 30 fibres were specifically placed in the bottom half of the specimen where most cracking was expected to occur, and the other sample contained fibres in a random distribution (RD) across the specimen, adding the fibres during the mixing process. The samples then underwent a three-point bending test, creating cracks of 0.3 mm in width using the CMOD clip gauge. It is interesting to note an elasticity within the k-SMP specimens upon unloading where the cracks closed to between 0.08 mm and 0.15 mm immediately upon release. This initial crack closure can be attributed to the elastic modulus of the PET fibres and was seen to stay in effect for the period after, closing the gaps further. After unloading, the samples were placed horizontally to rest for 24 h before subsequently being heated for 3 h, thus activating the k-SMPs. They were then left to air-cool for 12 h, providing opportunity for the cooling k-SMPs to retract further. Crack widths were subsequently measured. As was predicted by the researchers, the AD samples saw smaller cracks but larger proportionate differences in closure. Whereas the RD samples only averaged a crack closure of 45% across the front and back of two specimens, the AD samples averaged 77% crack closure. In addition, some of the AD data saw the k-SMPs completely close the gap as shown in It is clear that the orientation of the fibres has a strong impact on the performance of the tendons for the closure of cracks and therefore can act as a highly effective method for crack closure. Overall, it was concluded that the system was highly effective and creates opportunity for further research into the combination of these tendons with other self-healing technologies, such as micro-capsules and crystalline admixtures, in order to achieve total crack and stiffness remediation.The goal of the study was to compare, by means of a life cycle assessment (LCA), the potential environmental impacts of the kSMP concrete with those of conventional concrete. The analysis aims in particular to identify scenarios in which the kSMP concrete show advantages over conventional concrete. The assessments include the manufacturing of the two materials. However, use and end-of-life phases are currently out of scope due to lack of details at this stage.1 cubic metre of concrete (2400 kg) delivered to Cardiff.w/c) of 0.5.Concrete is produced in Rugby , using Portland cement Cem II 32.5 MPa, with a cement:sand:coarse aggregate ratio of 1:2:4 and water:cement ratio (Knotted SMP fibres are produced at University of Bradford (distance to Cardiff: 420 km) from commercial grade PET granulates (Dow Lighter C93), by melt extrusion followed by die drawing, both at 280 °C [In kSMP concrete, fibres are mixed with the concrete at a volume content of 0.82%, partially replacing coarse aggregate, as described in .Energy consumption associated with the concrete mixing and the activation system it is assumed negligible for this study.The following assumptions have been taken into account:3 of concrete. Since the purpose of the study is to compare two different materials, an attributional approach was adopted [The LCA was carried out according to ISO standards ,48. The  adopted ,50,51. I adopted . The lif adopted . The folIn accordance with the ISO standard 14044 (2006), normalization has been used to identify the impact categories most significant for the system under analysis. The LCA calculations have been performed under use of openLCA 1.0.3 , and theThe principal components of the two concretes assessed are listed in The environmental impacts for the kSMP concrete are compared with those of conventional concrete in Results for these three categories for the kSMP and conventional concrete are shown in The crack closure technologies presented contribute to the decarbonization of the concrete industry, by reducing the maintenance and repair and extending the service life of concrete structures. SMP products can be used in applications such as pre-cast concrete factories, where early age shrinkage cracking may compromise the quality and structural integrity of the elements. Another application is within the bridge construction sector, where early detection of damages and their repair is vital to ensure safety and long-term durability. Further, kSMP fibres could be used in engineering structures where regular inspections are challenging, such as oil-well cements and nuclear waste storage encapsulation. In both cases, the heat released by the pump fluid and the nuclear by-products, respectively, can be used to trigger the fibres shape memory effect.SMPs are being used to improve concrete structural integrity in a number of ways. High performance PET fibres have been produced by die-drawing and developed to provide high levels of shrinkage restraint force. SMP tubes, with complex molecular orientation fields, have been developed by mandrel drawing to withstand high compressive loads before activation and enable effective prestressed reinforcement devices.From the research reported in this paper, it may be concluded that the incorporation of PET-based SMP devices into cementitious structural elements provides an effective means of closing cracks that form after casting and curing.PET tendons alone are able to close cracks in plain mortar and concrete beams but have insufficient strength and stiffness to provide viable levels of prestressing for full-scale structural elements. The level of delayed prestressing afforded by these tendons is also insufficient to have a significant effect on reinforced concrete elements.Hybrid tendons, comprising bi-oriented PET tubes and a prestressed Kevlar core, are able to close serviceability-sized cracks in structural elements and provide substantial post-activation prestressing and reinforcement.Knotted PET SMP fibres used in cementitious composites provided a viable solution to effectively close cracks upon thermal activation in both aligned and random distribution, showing crack width reduction up to 77%.Future research on the SMP-based systems will involve the development of automated self-activation systems that are able to detect low damage levels as well as discrete cracks. Research is also needed to improve the efficiency of the tendons assemblies themselves, including enhancing the properties of the SMP sleeves used in the hybrid tendonsThe cradle to gate LCA highlights the dominant contributions of both cement and PET production to the environmental costs, with reference to the global warming and human toxicity environmental categories, which means that kSMP concrete was shown to have a higher environmental impact than conventional concrete. This result was expected, since the assessment did not include the whole life cycle costs. Thus, the benefits associated with reduced maintenance in terms of environmental costs were not reflected in the findings.


Advanced organic-inorganic materials-composites, nanocomposites, and hybrids with various compositions offer unique properties required for biomedical applications. One of the most promising inorganic (nano)additives are polyhedral oligomeric silsesquioxanes (POSS); their biocompatibility, non-toxicity, and phase separation ability that modifies the material porosity are fundamental properties required in modern biomedical applications. When incorporated, chemically or physically, into polyurethane matrices, they substantially change polymer properties, including mechanical properties, surface characteristics, and bioactivity. Hence, this review is dedicated to POSS-PU composites that have recently been developed for applications in the biomedical field. First, different modes of POSS incorporation into PU structure have been presented, then recent developments of PU/POSS hybrids as bio-active composites for scaffolds, cardiovascular stents, valves, and membranes, as well as in bio-imaging and cancer treatment, have been described. Finally, characterization and methods of modification routes of polyurethane-based materials with silsesquioxanes were presented. The aging population and increase in living standards has caused an increased need for biomaterials, which have seen rapid growth in recent decades . The resAmong those materials, nanocomposites have at least one dimension of the components ranging up to 100 nm. Such small sizes of nanostructures result in uncommon features of those materials because of prevailing surface properties over the material’s bulk properties. There are two main strategies for obtaining such materials: top-down and bottom-up. In the first case, the bulk material is split up to form nanomaterial, while in the second strategy, the nanomaterials are assembled from building blocks. Those building blocks, like molecules or clusters, are obtained from the bottom: grown from the elemental precursors. Nanostructures obtained by these techniques seem to be a key in producing nature mimicking biomaterials since the natural structures are hierarchical. In this field, the polymeric nanocomposites that contain inorganic nanofillers combine beneficial features of both phases, and even synergistic effects may be observed. For example, adding a nanofiller may result in better thermal and chemical resistance, mechanical properties , as well as electrical and magnetic properties .On the other hand, the polymer matrix provides processability, lightweight, and flexibility. The key to this approach’s success is to keep a good interaction between nano and polymeric phases resulting in good homogeneity of a dispersed phase in the continuous phase. Therefore, there is tremendous interest in nanocomposites in which covalent bonds are formed between the phases, resulting in homogeneity and stability of the material, e.g., migration of the filler is prohibited. On the other hand, covalent bonding of the phases with low compatibility results in phase-separated, self-assembling materials showing biocompatible and biostable properties . When orOne class of the finest well-defined nanoparticles are polyhedral oligomeric silsesquioxanes (POSS), considered the tiniest glass nanoparticles. Although they were first synthesized in the 1870s, they are a new, rediscovered generation of zero-dimensional nanomaterials, with the structure resembling a cage in which corners are silicon atoms bound with oxygen bridges . Their ePolyurethanes (PUs) are a group of polymers that possess urethane bonds in the main chain. POSS has been used in polyurethanes for multiple applications. Noteworthy, POSS-PU nanocomposites are known to have higher thermal stability ,29,30,31In medicine, PU are important materials since they can be simultaneously elastic and thermoplastic (thermoplastic polyurethane–TPU) or porous (rigid and flexible foams); by choosing the amount and type from a variety of polyols and isocyanates, they have the potential of tailorable properties. Moreover, they show good blood compatibility and may be a biostable and biodegradable component in medical applications due to a wealth of possible compositions. The ease of properties tailoring is crucial as the material has to fit the properties of surrounding tissue. For instance, PU biomaterials in the form of foams can replace soft tissues. However, as they are traditionally obtained from the most reactive aromatic isocyanates, they are believed to leak carcinogenic 2,4-toluenediamine (TDA) ,42. EvenHence, the exciting idea in this field is to produce non-isocyanate polyurethane (NIPU) obtained most promisingly from cyclic carbonates reacting with amines ,46,47. DThis review is dedicated to POSS-PU composites and comprises four major parts. In the In POSS molecules, functionalization is possible at the silicon corners of the cage. For example, the most commonly used cube-shaped T8 system contains eight silicon corners to which any organic group can be attached. Functionalization is possible at the stage of molecule formation or later through the hydrosilylation reaction. In the case of polyurethanes, functional groups enabling dissolution in the matrix are often used, e.g., tert-butyl, phenyl, cyclohexane, and various types of functional groups, such as isocyanates, amines, epoxides, and alcohols. POSS can be introduced in various ways into the system, which affects the macromolecular architecture and the degree of homogenization. For example, POSS molecules can be grafted on macromolecules, introduced as pendant groups, telomerised, cross-linked, and arranged in nets .Rigidly bonded POSS are usually those with partially condensated cages and functional groups attached to both sides of the cage B POSS in2/N2 and CO2/N2 gasses [The open-cage POSS is used in another approach in which a reaction between isocyanate and silanol groups occurs. For instance, three-functional open cage POSS (trisilanol) was used to react with HDI; DBTL was applied as a catalyst . Trisila2 gasses .An exciting class of silsesquioxanes are double-decker silsesquioxanes (DDSQs) that possess two reactive groups on each side of the molecule and two siloxane “decks” parallel to them. Recently, DDSQ with two hydroxyl groups on both sides has been reacted with HDI and PCL to form a hybrid material characterized by increased thermal stability, surface hydrophobicity, and lipophobicity ,64,65.POSS can be attached as pendant groups on the polymer chains by using a difunctional POSS substituted with one chain containing two reactive functional groups ,66,67,68Grafting is a method in which a previously obtained polymeric chain or surface is grafted with POSS, usually to add functionalization or increase compatibility when blending with another polymer  Figure D. For exCrosslinking is a network formation process, and POSS with multiple functionalities can act as efficient crosslinking agents E. Hence,Chain termination/telomerization with POSS facilitates phase separation effects resulting in improved hydrophobicity and contact angle increase  Figure F. MoreovThe high functionality of POSS can be used to create polymer networks, in which POSS cages act as enhancements G. POSS iIn addition to bulk polymerization, solvent polymerization is of interest, where POSS can be introduced in a solution, facilitating its homogenization. Since copolymerization is completed, the polymer precipitates from the solvent, or the solvent evaporates by the exo-energetic chain growth effect or by reducing the pressure . ConductBlending is a physical process when POSS is mixed with the polymer without a chemical reaction. POSS is functionalized only with the nonreactive groups that enhance compatibilization between the POSS and a polymer H. This ww/w = 1:2) was electrospun with 0.8 mL/h extruding speed, at 20 kV, and with 20 cm distance between tip and collector. Only 2% of POSS in the obtained membrane had significantly increased contact angle, inhibited bacteria growth, and decreased platelet and protein adsorption. The results are promising; however, cytotoxicity has not been tested. Blood biocompatible membranes can be obtained by electrospinning of POSS-PU copolymer. The homogeneous mixture of open cage trisilanolisobutyl-POSS (TSI-POSS) in PTMG was reacted at 110 °C with MDI and chain extended with BDO. The 12% of POSS-PU solution in DMF/THF  are one of the most used cationic disinfectants, mainly because they can kill the versatility of microorganisms like fungi, bacteria, and algae . MoreoveA similar approach was applied by Chawla et al. in which two types of POSS/polyurethane copolymers have been used to support each other in wound healing applications. The first copolymer was biodegradable POSS-PCL (POSS incorporated into poly(caprolactone-urea)urethane) containing poly(hexamethylene carbonate), which was protected by the second copolymer, biostable POSS-PCU (POSS incorporated into poly(carbonate-urea)urethane). The outer layer of POSS-PCL was designed to protect the inner, biodegradable POSS-PCU, and thus it was impregnated with silver as an antibacterial agent. As mentioned earlier, both copolymers are known for their excellent microporous topography and great distribution of pores that allowed cellularization with adipose tissue-derived stem cells (ADSCs) within a few days, making the composition well-suited in wound healing applications. Silver was applied as nanoparticles, and authors declared no cytotoxicity of the composite, although no immobilization of silver was secured. The first scientific reports about silver germicide properties come from the XIXth century, but they are known from ancient times. Silver nanoparticles are of much interest because of their significant antimicrobial activity ,112,113.Not only silver is used as a metallic antimicrobial agent. Copper is also known for centuries and in ancient times was used to treat wounds and sterilize water. Cu ions sterilization properties are due to the generation of hydroxyl radicals that oxidize bacterial proteins, lipids, DNA, and other (macro)molecules . NoteworPOSS high functionality, self-assembly tendencies, and biocompatibility can meet in drug delivery applications . POSS coBy the year 2015, cancer had caused 15.7% of deaths . POSS coIn biomaterials, POSS plays an essential role as a bio-compatibilizer due to bearing multiple Si-O bonds. Molecules containing this bond, especially when combined with hydrocarbons, are usually biocompatible because of Si-O bond-induced chemical stability and surface properties (low surface energy and hydrophobicity) . In addiPolyurethane is widely used in the field of biomaterials, especially for blood-compatible materials. The applications range from catheters to artificial hearts, and polyurethane is the third most commonly used polymer for vascular surgery ,138,139.Here is where POSS can come into the light. Because of its variable surface tension, the use of POSS in those materials resulted in platelet and fibrin repulsion and significantly increased thromboresistance without elasticity mismatch and intimal hyperplasia effects associated with materials containing silicon ,141. ThePOSS-PCU was synthesized from MDI, trans-cyclohexanechloroydrinisobutyl POSS, and poly(carbonateurea) glycol, and a mixture of 40:1 (by wt.) ethylenediamine: diethylamine as a chain extender . In the ® porous polyethylene have been investigated by various methods. Protein adsorption, Human Dermal Fibroblast seeding, and assessment of their morphology, adhesion, growth, and secretion of collagen and VEGF have been performed. Furthermore, seeding of macrophage cell cultures, assessing their adhesion and growth, and immune response in vitro and in vivo analysis on laboratory mice were conducted. Protein adsorption, vital for cell adhesion onto the surface [Most of the further investigations on POSS-PCU were about modification techniques. Auspicious results have been reported recently concerning argon plasma surface modification of POSS-PCU ,145. The surface ,147. Hig surface ,101. In  surface , argon p surface ,150. When the material is not being rejected, one can focus on the mechanical properties of the implant. The synthetic scaffolds and implants are often designed as stronger and stiffer than the surrounding tissues due to the focus on optimizing mechanical stability without paying attention to adapting the implant in an organism-environment. Due to the apparent difficulties with the diversity of living organisms within even one species, most tests are performed in vitro, not in vivo. Nevertheless, a mismatch of the mechanical scaffold properties in living tissue can result in remodeling of the host tissue, bones becoming less dense and weaker, cartilages remodeling its stiffness or formation of fibrous scar ,152,153.1k) or D,L-lactide ((PLCL)1k), copolymerized with ε-caprolactone and butanediol [Biodegradable PU-POSS materials are usually composed of biodegradable polyol, mostly polycaprolactone diol (PCL). An example can be a polymer synthesized using HDI (hexamethylene diisocyanate) and PHI-POSS . Additionally, PCL was used as a biodegradable diol, synthesized from either glycolide ((PGCL)tanediol . The reatanediol .The requirements for tissue engineering for the vascular grafts have been described in detail by Boffito et al. . The impIn detail, the responsibility of the implant patency is mainly on the side of a suitable charge, energy, wettability, and topography ,159,160.In another way, the foreign body response reaction begins with protein adsorption due to exposure to the body fluids. The protein layer is formed on the implant surface, which attracts the cells of the immune system. The macrophages recognize it as a foreign invader and make attempts to devour it. Because the implant size is much bigger than the cells themselves, they fuse to form foreign-body giant cells (FBGCs) and release signals to attract the fibroblasts ,169. To Bare metal stents (BMS),Drug-eluting stents (DES),Coated stents,Graft-covered stents.Four main types of endovascular stents can be distinguished :Bare metMay be folded or compressed for efficient delivery,Have high flexibility for easy maneuvers during delivery and deployment,Possess predefined expandability rates and strength with negligible recoil after implantation,Low deployment pressure,High burst resistance (>500 mm Hg),2 min−1 at 120 mm Hg),Low water permeability (1 mL/cmControllable with the use of magnetic resonance imaging (MRI),Haemocompatibile, not causing allergenic reactions,Modified biomimetic surface with biomimetic peptides, antibodies and growth factors, nanomaterials.Despite significant advances in the stents field, the main problem with their development is still thrombosis/restenosis response , which aThe effortlessly tailored properties of polyurethanes and biocompatibility of PTFE have decided their general use in this field. However, their main drawback is low oxidation resistance. To overcome these problems, POSS copolymer with poly(carbonate-urea) was synthesized, which had fruited with a family of non-biodegradable and bioabsorbable polymers that meet the essential criteria in the development of covered stents. This composite is biocompatible, provides excellent endothelialization support, is non-toxic, causes no inflammatory response, being simultaneously strong and durable . FurtherMoreover, POSS can be further modified by incorporating other functional groups that modify the copolymer chain or surface. The functionalization may also be done from the metallic stent site for a better junction between polymer and metal. It is beneficial to apply polymer by spray technique, which ensures better folding and flexibility of the stent. Electrospray or ultrasonic sprayed film must cling well because the exposed bare metallic stent surface has thrombogenic potential, mainly by releasing Ni ions .The surface modification technique may be based on anodization of NiTi alloy in sodium nitrate MetOH solution and 1-h heat treatment at 600 °C. The resulting titanium oxide coating is then subjected to sodium hydroxide to saturate the surface of the oxide with OH groups entirely and then silanized by 3-aminopropyltriethoxysilane in acetic acid for 15–60 min at room temperature. Then, cross-linking may be done at the elevated temperature . As a result, the metal surface is covered with Ti-O-Si bonds with the aminopropyl end that can react with PCU/POSS after primed with the prepolymer and covered with PCU/POSS hybrids using ultrasonic atomizing spraying technique. The chemistry of the process is shown in Silanisation can also be done for other types of BMS, for example, the CoCr type. An important parameter is the thicknesses of the polymer covering membranes. It was found that the optimal thickness in the case of POSS/PCU is 20 µm; above this value, the stent recoil percentage is drastically increasing .The second approach to functionalization are biomimetic surfaces that are getting more and more attention in the new compositions for stent coating. A remarkable advance was the composites’ ability to capture anti-CD34, the epithelial progenitor cells (EPC) antibodies, crucial for endothelialization . AuthorsPU-POSS composites with their ability to separate into POSS microdomains were utilized in another type of stents, namely polymeric shape-memory stents. Their ability to self-expand in the pre-set temperatures could be applied in stents expanding in the body temperature range. Those materials contain a two-phase shape transition. In the first phase, they may be fixed in a temporary shape, while in the second phase, the polymer is stimulated  to recover its permanent shape. The thermally-induced shape memory polymers are obtained by forming cross-linked networks while maintaining the ease of chain conformation between adjacent cross-linking sites . Thus, sOn the other hand, most polymers gradually lose mechanical properties after application due to fatigue failure ,181. Eff7 pulses. X-ray, microscopic, and chemical investigations were performed to follow the signs of calcium deposition. The chemical analysis evidenced that calcification was significantly reduced in POSS-PCU contrary to pure polyurethane and bovine pericardium [2 plasma treatment, which formed hydroperoxide groups on the surface, and OVS was able to chemically bond with them. Bonding was visible as a reduction of 1380 cm−1 band attributed via FTIR-ATR analysis to -OOH groups. The presence of HNCT had a synergistic effect on OVS grafting since EDS maps showed its higher concentration on PU/HNCT than on pristine PU. Plasma-induced POSS grafting had expanded the average surface roughness substantially and wetting angle up to 165°, which resulted in lower calcification and lower platelet adhesion; however, in the last case, plasma treating alone seemed to be essential. Since the 1950s, polyurethane valves have been in use. However, they lack long-term durability . The maiicardium . The keyicardium ,187. GraAnother application of POSS-PCU are synthetic nerve conduits. They were obtained by subsidizing PCU with the RGD peptide (arginine-glycine-aspartamic acid)  and are Two types of POSS: octa(3-hydroxy-3-methylbutyldimethylsiloxy) POSS (OCTA-POSS) and 1,2-propane-dioliso-butyl POSS (PHI-POSS), were used to modify flexible polyurethane foams as potentially active bone scaffolds. The foams were produced with aliphatic hexamethylene diisocyanate (HDI), which is less toxic than MDI, and a mixture of two polyetherols: sorbitol-based and glycerine based one, with POSS loadings varying from 0 to 15 wt% and water as a blowing agent. The compressive strength of the foams at 40% deformation was highest for small amounts of POSS, deteriorating with further POSS loadings and resulting in POSS crystallization. Crystallization was visible via WAXD in the case of PHI-POSS, and the compression strength of those samples was 150% higher than reference in lowest loading and 75% lower in highest loading. Despite no evidence of crystallization, OCTA-POSS also followed this trend, reaching ~200% and 150% increase for lowest and highest loadings. After four-week incubation in simulated body fluid (SBF), scanning electron microscopy showed numerous spherical hydroxyapatite (HAp) granules that were directly size- and amount-dependent on POSS concentration in PU matrix. Energy-dispersive X-ray spectroscopy revealed a Ca/P ratio varying from 1.54 to 1.63, indicating the presence of well-defined HAp. After preparation, the adherent established cell line of human osteoblast U-2 was placed on materials and cultured for 24, 48, and 72 h in a laminar flow chamber. The cell cycle and apoptosis with Annexin V were evaluated. Materials negatively affected the cell viability, but with higher loadings of POSS, the cytotoxicity was reduced, as a noticeably smaller percentage of dead cells was observed in the case of modified foams compared to reference . POSS-PCU-PCL was also used as a porous scaffold for liver restoration . The por3 particles (100–150 µm in diameter) and NaHCO3 with DMAC were extracted with water. The obtained product was seeded with human bone marrow-derived mesenchymal stem cells (BM-MSC) at 200,000 cells/m2 in Bronchial Epithelial Growth Medium (BEGM) incubated for 48 h. Then the medium was changed into Minimal Essential Medium (αMEM), and primary human airway epithelial cells were seeded with density 1.5 × 106/m2 and, after 72 h, orthotopically implanted in place of a previously prepared laryngeal defect. One from eight pigs had expectorated its implant and was terminated early–after two weeks. Endoscopy was performed at weeks 1, 2, 4, and 8 showing a smooth appearance suggesting mucosal regeneration, and mucosal brushing showed cells with epithelial cell morphology and no phenotypically-human cells (beyond four weeks). Full extrusion of implants was complete at the time of termination (8 weeks) [One of the most challenging procedures is replacing the upper airway epithelium with exogenous cells . POSS-PC8 weeks) . The sucA solvent-melt method can be used to produce artificial meniscus material. It was synthesized by using monofunctional POSS that was telomerising polyurethane chain. It was found that polyurethane chains capped with POSS have better mechanical properties, oxidation, and calcification resistance. Furthermore, the cell proliferation analysis done by fluorescence tracking showed that many cell cultures were proliferating in-vitro, and fusing proceeded more quickly than in the control sample. The POSS-capped polyurethane scaffold showed well in vivo biocompatibility; however, fixing the composite in the meniscus prosthesis is still challenging .Adhesion strength is an essential factor of covered stents. The broadly used nitinol stents were entirely covered to prevent Ni ions from leaching, facilitating thrombus formation. The proposed technique is based on classical tensometer measurement in which one handle is holding a stripe, manually peeled off from the polymeric coating, and another clamping the stent with an initial distance of 10 mm between the grips. In earlier studies, a tensometer equipped with a 500 N load cell was used to measure the adhesion, and the procedure was based on the ASTM D413 standard, which gives recommendations on how to determine the force per unit width required to separate a rubber layer from a flexible substrate such as fabric, fiber, wire, or sheet metal . The tesθ angles for two different liquids like water (highly polar liquid) and diiodomethane (highly dispersive liquid). A syringe pump is used to dispense the sessile drop through a flat tip needle. The sessile drop must be small to reduce the gravitation effect, and typically it has a volume of a few μL. The wetting angle is measured via digital analysis software [θ are considered hydrophobic, and values <90° θ are considered hydrophilic. The wetting angle is an essential property of biomaterials. In the case of biostable materials, it should be high to enhance its stability, while in the case of biodegradable materials, it should be low to encourage cells proliferation. Several methods measure contact angle and subsequent surface free energy, such as static and dynamic sessile drop methods, captive bubble angles, pendant drop methods, various Wilhelmy methods, and finally, capillary rise method. Sessile drop is widespread as it is simple and does not need advanced equipment. Surface-free energy and its dispersive and polar components may be measured by comparing software ,174. TheThe porosity is thought to be an essential factor affecting cell migration into the biomaterial . TherefoMicroscopies like SEM or AFM usually measure surface porosity. In the SEM technique, graphical analysis software shall measure the surface pore size distributions to obtain numeric data. For AFM, the roughness may be quantified by the deviation of the real measured surface from the area measured . Macrophage polarization from macrophage phenotype M1 to a macrophage phenotype M2 shows how a living tissue reacts to the presence of the implanted biomaterial. M1 macrophages produce proinflammatory cytokines, participate in phagocytosis, and, in the case of biomaterials, produce FBGCs. M2 macrophages facilitate proliferation and induce collagen production, associated with wound healing and acceptance of the biomaterial. The assessments of those macrophage types can be done by immunofluorescent staining to detect the presence of the macrophage capillary markers. CD86 and CD68 are the markers of macrophage M1 and CD163 for the M2; also, T-cells may be measured by CD3/CD4. A confocal microscope can be used to capture images and stained cells counted by image analysis software . Plasma is defined as a gas in an ionized state . Among vThe method is helpful in the fabrication of covered stents . The metAn advantageous technique in scaffold production is TIPS  . This te3D/TIPSRecent advances in this technique include :3D/TIPSIt is a combination of 3D print with TIPS that can produce a multilevel, hierarchical structure of extracellular matrix (ECM). 3D printing technology is used to obtain a preform from a polymer that is easy to extract (like PEG), which is immersed in a solution of another polymer that is then thermally separated. The solvent is extracted by another solvent, as well as the first polymer preform. The obtained macro/micro scaffold is much-resembling nature .TIPS and electrospinningElectrospinning uses an electric current to draw charged threads from the polymer-solvent system or polymer melt. By using electrospinning, nanofibers can be produced, which may be used as a preform in the TIPS technique. By combining these techniques, a network of interconnected channels can be produced resembling the vasculature of scaffold structure .TIPS and porogen leaching3, sugar) is mixed with the polymer and then extracted, leaving pores with predefined size and morphology [This technique utilizes the well-known porogen leaching technique in which usually water-soluble salt urethane, and its derivatives containing various concentrations of polycaprolactone as a biodegradable component of the main backbone. Those matrices are either copolymerized with trans-cyclohexanediolisobutyl-POSS or telomerised with trans-cyclohexanechloroydrinisobutyl POSS. This composition went into further commercial development under the name UCL-NanoBioNevertheless, attempts are made to obtain other biocompatible matrices, and some of them appear to have great potential. On the POSS side, numerous research efforts are dedicated to chemical functionalization, including chemical bonding of drug molecules and utilization of self-assembly effects. An inspiring way of further topic exploration may be the incorporation of POSS into NIPU matrices, primarily because of their natural hydrophilicity that does not have to be induced by further modifications.


Antibiotic resistance (AR) is indisputably a major health threat which has drawn much attention in recent years. In particular, the gut microbiome has been shown to act as a pool of AR genes, potentially available to be transferred to opportunistic pathogens. Herein, we investigated for the first time changes in the human gut resistome during aging, up to extreme longevity, by analyzing shotgun metagenomics data of fecal samples from a geographically defined cohort of 62 urban individuals, stratified into four age groups: young adults, elderly, centenarians, and semisupercentenarians, i.e., individuals aged up to 109 years. According to our findings, some AR genes are similarly represented in all subjects regardless of age, potentially forming part of the core resistome. Interestingly, aging was found to be associated with a higher burden of some AR genes, including especially proteobacterial genes encoding multidrug efflux pumps. Our results warn of possible health implications and pave the way for further investigations aimed at containing AR accumulation, with the ultimate goal of promoting healthy aging.IMPORTANCE Antibiotic resistance is widespread among different ecosystems, and in humans it plays a key role in shaping the composition of the gut microbiota, enhancing the ecological fitness of certain bacterial populations when exposed to antibiotics. A considerable component of the definition of healthy aging and longevity is associated with the structure of the gut microbiota, and, in this regard, the presence of antibiotic-resistant bacteria is critical to many pathologies that come about with aging. However, the structure of the resistome has not yet been sufficiently elucidated. Here, we show distinct antibiotic resistance assets and specific microbial consortia characterizing the human gut resistome through aging. Rates of infection with antibiotic-resistant microorganisms continue to rise worldwide . In ItalImportant evidence of the impact of antibiotic misuse/abuse and environmental exposure on the development of AR was provided by the comparison of the gut resistome  of Western populations with that of traditional communities , 8. Base12–The relevance of antibiotic-resistant gut bacteria as an immediate and long-lasting threat to human health is well recognized, especially in compromised individuals, such as preterm infants receiving early-life antibiotics , elderlyIn an attempt to bridge this gap, here we profiled the human gut resistome of an exceptional cohort of semisupercentenarians, i.e., extremely long-lived individuals over the age of 105, compared to those of young adults, elderly, and centenarians, from a specific geographic area . Specifically, we characterized the type and target of resistance and related bacterial taxa in fecal samples from 62 individuals falling within the four age groups mentioned above. In addition to providing a fine characterization of antibiotic resistance within the gut microbiome of subjects at distinct times of life, our study emphasizes a progressive age-related burden in AR genes assigned to potential pathobionts.We previously identified considerable taxonomic and functional variability in the gut microbiome structure of a geographically defined cohort of 62 urban individuals, including 11 young adults , 13 younger elderly individuals , 15 centenarians , and 23 semisupercentenarians  . Here, wP = 0.0015 and 0.001, respectively; permutation test with pseudo-F ratios, vegan R package)  and Ruminococcaceae , as well as Bifidobacteriaceae  , Eubacteriaceae , Prevotellaceae , and Veillonellaceae , along with an increase in AR reads assigned to Enterobacteriaceae    based on the Bray-Curtis distance between the taxonomic profiles of the gut resistome at both the family and genus levels (package)  and d. T5% in S) . Compare = 0.01) . The age≤ 0.029) .10.1128/mSphere.00691-21.1FIG S1Bacteroidetes during aging. Box plots showing the relative abundance distribution of AR reads assigned to Bacteroidetes in the gut resistomes of young adults (Y), younger elderly individuals (E), centenarians (C), and semisupercentenarians (S). An age-related decrease was found . Download FIG S1, EPS file, 0.06 MB.Relative abundance of AR reads assigned to the phylum Copyright © 2021 Tavella et al.2021Tavella et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Bifidobacterium , Faecalibacterium , and Collinsella , but with a decreased proportion of Faecalibacterium-assigned AR reads occurring with aging   and Ruminococcus , which are other typically health-associated, short-chain fatty acid-producing taxa  and Megasphaera . On the other hand, the fecal resistome of extremely long-lived people is enriched in AR reads assigned to Eggerthella     is essentially dominated by  = 0.02)  and f. Cing taxa , as well = 0.01) . A linea ≤ 0.05) . When ex ratios) , thus su10.1128/mSphere.00691-21.2FIG S2P values were corrected with the Bonferroni method. CPM, counts per million. Download FIG S2, EPS file, 0.4 MB.Association of age with the taxonomic features of the gut resistome. Scatterplots and Poisson regression analysis of AR reads assigned at the family (a) and genus (b) level as age advances. Only the taxa that emerged in the comparison between age groups  were conCopyright © 2021 Tavella et al.2021Tavella et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00691-21.3FIG S3P = 0.45 and 0.47 for family- and genus-level PCoA, respectively; permutation test with pseudo-F ratios). Download FIG S3, EPS file, 0.1 MB.Impact of gender on the compositional structure of the gut resistome. Principal-coordinate analysis of Bray-Curtis dissimilarities between the family-level (a) and genus-level (b) profiles of the gut resistomes of the study subjects, stratified by gender . No significant separation was found  , 1 encodes a quinolone resistance protein (mfd), and 1 encodes an isoleucyl-tRNA synthetase (ileS) conferring resistance to mupirocin.Consistent with taxonomic data, the Bray-Curtis PCoA on protein-level resistome profiles provides evidence of an age-related trajectory ( ratios) , suggest ratios) . In part2 fold changes of ≤−2 and ≥2). Twenty-nine of these discriminating ARDs were annotated as coding for efflux pumps, 7 as antibiotic inactivating, 2 as antibiotic target modifying, and 1 as target protecting. The full list of age group-specific ARDs, including a description of their mechanisms, is reported in leuO) and multidrug, particularly bcr, emrD, emrY, mdfA, mdtG, mdtL, robA, and tolC . Seven multidrug ARDs are specifically discriminatory for semisupercentenarians  (P ≤ 0.008).A total of 39 age group-specific ARDs were next identified, defined as AR determinants that were differentially represented in at least one comparison between two age groups , the name of the gene, the family, the resistance mechanism, and the antibiotic to which the resistance is conferred are reported. The annotation was manually curated from the Antibiotic Resistant Ontology (ARO) from the CARD database. Download Table S2, DOCX file, 0.01 MB.Age group-specific antibiotic resistance determinants. For each antibiotic resistance determinant (ARD), identified by the Wald test  on differences in mean abundance of antibiotic resistance determinants (ARDs) between age groups. For each ARD, the mean abundance, the logCopyright © 2021 Tavella et al.2021Tavella et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the rphB) and tetracycline (tcr3 and tetD) (P ≤ 0.02). On the other hand, compared to that of semisupercentenarians, the resistome of young adults was enriched in ARDs for beta-lactam antibiotics , whereas that of elderly subjects was enriched in ARDs for macrolide-lincosamide-streptogramin (ermB and ermF) (P ≤ 0.05).Furthermore, the semisupercentenarian group showed higher levels of ARDs conferring resistance to rifampin , validated by at least two of the three methods presented   . The mai10.1128/mSphere.00691-21.4FIG S4x axis while the normalized relative frequency of the ARD is on the y axis. Download FIG S4, PDF file, 2.8 MB.Trend plots showing the fitted Poisson regression model between age as a continuous variable and antibiotic resistance determinants. Plots, computed with the ShotgunFunctionalizeR package, are shown for the 25 antibiotic resistance determinants (ARDs) found to be significantly associated with age . For eacCopyright © 2021 Tavella et al.2021Tavella et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00691-21.9TABLE S4P value, and the antibiotic to which the resistance is conferred are reported. P values were corrected with the Bonferroni method. Y, young adults; E, younger elderly individuals; C, centenarians; S, semisupercentenarians. Download Table S4, DOCX file, 0.02 MB.Validation of age group-specific antibiotic resistance determinants by overdispersed Poisson generalized linear model analysis. For each antibiotic resistance determinant (ARD), the comparison between age groups, the coefficient, the direction of the coefficient (and whether the DESeq2 datum is confirmed or not), the Copyright © 2021 Tavella et al.2021Tavella et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00691-21.10TABLE S5P value corrected with the Bonferroni method are reported. Download Table S5, DOCX file, 0.01 MB.Results of regression of antibiotic resistance determinants and age as a continuous variable, using the overdispersed Poisson generalized linear model. The ShotgunFunctionalizeR R package and testGeneFamilies.regression function were used. For each antibiotic resistance determinant (ARD), the coefficient and the Copyright © 2021 Tavella et al.2021Tavella et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Proteobacteria  and a lower contribution of ARDs from Bacteroidetes  in semisupercentenarians than in younger adults. It is worth noting that 46.3% of proteobacterial ARDs encode multidrug efflux pumps of the species Escherichia coli. On the other hand, the most represented ARDs in the fecal resistome of young adults are beta-lactamases from Bacteroidetes species .When looking at the taxonomic classification of these ARDs, we found that they were differently assigned even at the phylum level, depending on the age group , hintingarnD) with antibiotic target protection mechanism (ARO:0001003). The AR protein families linked to E. coli are the major facilitator superfamily (MFS), accounting for 91.3% of E. coli links, and the resistance-nodulation-cell division (RND) antibiotic efflux pump family (4.3% for acrD). E. coli was found to have the highest diversity in terms of ARDs, with links also to determinants conferring resistance to sulfonamide and glycopeptide drugs. On the other hand, Bacteroidetes species, including Alistipes (from the family Rikenellaceae), are linked to beta-lactamases, with Bl2e_cepa, cblA-1 and OXA-34 accounting for 80% of the nodes, and to the RND antibiotic efflux pump family (mexW), accounting for the remaining 20%. The main results were validated by performing the correlation analysis with the SparCC method, using the 26 ARDs validated by at least two of the three methods in our ensemble approach . Our cohort was recruited from the Emilia Romagna region , and its gut microbiome was previously characterized in terms of taxonomic and functional structure , 20.Lachnospiraceae and Ruminococcaceae, along with Bifidobacteriaceae. This could be the result of extensive microorganism-microorganism cross talk within the gut microbiome, with the spread of AR genes via horizontal gene transfer, potentially fueled by antibiotic exposure. On the other hand, in line with gut microbiota data   , while tIn light of the increased vulnerability of older people to infectious diseases , the emeBacteroidetes members. As previously discussed, genes conferring beta-lactam resistance are frequently present in Bacteroides spp. and among the most abundant AR genes in the human gut microbiome , sheds some light on the possible trajectory of the human intestinal resistome during aging and draws attention to the potential age-related accumulation of AR genes with possibly severe repercussions on human health. Future studies in larger cohorts will ideally need to sample the same subjects over time. In any case, information on previous antibiotic exposure and medical history, over the widest possible window of time, will need to be included in an attempt to provide mechanistic explanations. Other potential confounders, such as gender, lifestyle, and geography, will need be considered to draw unambiguous conclusions about aging and resistomes. In addition to stressing the relevance of resistome surveys for more effective therapies, our results may support campaigns aimed at changing human behavior, i.e., reducing the use and abuse of antibiotics, with the ultimate goal of containing the spread of AR, thus supporting healthy aging.Here, we analyzed shotgun metagenomics reads of 62 fecal samples from 62 Italian subjects, generated in a previous study . All sub10.1128/mSphere.00691-21.6TABLE S1Table S1, DOCX file, 0.02 MB.Metadata available for the study cohort. For each subject, ID, age (and group), gender, and use of prescribed drugs in the month prior to fecal sampling, with reference to antibiotic, cardiovascular, antihypertensive, hypoglycemic and hypolipidemic therapy, are reported. Y, young adults; E, younger elderly individuals; C, centenarians; S, semisupercentenarians. Download Copyright © 2021 Tavella et al.2021Tavella et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the https://www.ncbi.nlm.nih.gov/bioproject/PRJNA553191).For DNA extraction and library preparation, see reference in silico depleted of host DNA, using the NCBI Homo sapiens assembly 19 as a reference genome, identified with bmtagger software  and removed with the BBMap tool (http://sourceforge.net/projects/bbmap/). Raw reads were processed with Trimmomatic , was computed with the Ward linkage method over the Euclidean distances (pheatmap package) (Age group-specific antibiotic resistance determinants (ARDs) were identified by the Wald test as implemented in the DEseq2 package  in R, wipackage) . An overP values of 0.001 (Bonferroni method), was visualized as a network    and reta. 0.9.2) . Correlaretained .