Since most starting materials for tissue engineering are in powder form, using powder-based additive manufacturing methods is attractive and practical. The principal point of employing additive manufacturing (AM) systems is to fabricate parts with arbitrary geometrical complexity with relatively minimal tooling cost and time. Selective laser sintering (SLS) and inkjet 3D printing (3DP) are two powerful and versatile AM techniques which are applicable to powder-based material systems. Hence, the latest state of knowledge available on the use of AM powder-based techniques in tissue engineering and their effect on mechanical and biological properties of fabricated tissues and scaffolds must be updated. Determining the effective setup of parameters, developing improved biocompatible/bioactive materials, and improving the mechanical/biological properties of laser sintered and 3D printed tissues are the three main concerns which have been investigated in this article. In eachBiomedical applications, e.g., tissues, scaffolds, and fixation devices, have specific aspects of fabrication which should be considered. For biomedical applications, the use of these AM methods without rigid support structures is strongly recommended . In suppAM approaches, particularly 3DP and SLS, are simple and adaptable to using a broad range of powders to produce porous ceramics, polymers, and metal-based tissues , 8. To ein vitro, in vivo and apatite layer formation) conducted on the fabricated tissues and scaffolds are presented and discussed, as well as clinical works regarding fabricated objects.In this article, the working principle of SLS and inkjet 3DP and modifications of these methods are reviewed. Materials used in SLS and inkjet 3DP and optimization of the effective parameters of these two powder-based AM techniques for the fabrication of useful bone tissues and scaffolds are highlighted. Biological tests  occurs. It can be observed in amorphous material. Melting does not occur until the polymer reaches a higher temperature, Tm. Below Tg, the polymer is in the glass state and the molecular motion along the chain is frozen. When the temperature rises from Tg to approximately (Tg + 30 K), the molecular motion increases, causing the modulus to drop. Just above Tg, the polymer behaves like a highly viscous liquid in which the chains are all tangled up with their neighbors [The most important characteristics that determine the application of thermoplastic polymers are the glass transition temperature, eighbors , 37.Tg) below or close to room temperature (−100 to 50 °C) and a melting temperature (Tm) above 100 °C (between 100 and 400 °C) at which a considerable volume change occurs. On the other hand, amorphous polymers do not have a characteristic melting temperature range [Tg of ∼100 °C, above which the material will progressively evolve to a leathery, rubbery, and finally liquid state as the temperature increases, with no obvious transitions [Tg and Tm depend directly on molecular weight. This is why a different setup is needed to run an SLS system for different thermoplastic materials.It has been also reported that a majority of semi-crystalline polymers have a glass transition temperature  is a bioactive ceramic that has been explored for tissue engineering applications over the last two decades [10(PO4)6(OH)2). HA is a calcium phosphate ceramic (CP) material that is biocompatible and bioactive due to its similarity to the mineral constituents of human bone and teeth [Agglomeration of powders is a concern with using slurry-based SLS. An effective way to avoid agglomeration during laser sintering may be to process at a lower scanning speed or to employ a surfactant in a very low concentration . Using a decades –55. Many decades . Anothernd teeth , 58. Nannd teeth , 58.The indirect method uses polymers as a binder with ceramic powders as the main matrix and involves the melting of sacrificial organic polymer to obtain a green part. The green parts are subsequently sintered to produce the final porous ceramic parts . Even thTm with SLS heating it in just a small window, i.e., the temperature window between the onset of polymer melting during heating and crystallization during cooling [It has been confirmed that semi-crystalline polymers are preferred over amorphous polymers for use as the binder phase due to their higher density compared with amorphous polymers . However cooling , 60. AftFabrication of scaffolds from bioactive glass materials using the indirect method has been reported. Bioactive glass materials with different compositions, e.g., 45S5, 58 S, and 13–93, can be used as scaffold materials , 61, 62.2.1.3.Because metals possess excellent compressive strengths and also high fatigue resistance, porous metallic scaffolds such as titanium (Ti) and tantalum (Ta) and biocompatible alloys such as CoCr and nitinol have been proposed as bone replacement materials, but unlike bioactive ceramics or biocompatible polymeric scaffolds, biomolecules cannot be integrated into metallic scaffolds. The lack of degradability of metallic implants restricts the use of these kinds of scaffolds. The main concern with embedding metallic scaffolds is metal ion release into body fluid, leading to sarcoma. Coating the surface of metallic scaffolds with bioactive ceramics such as HA or CS or using surface finishing methods is highly recommended to improve the biological properties of metallic scaffolds .One category of SLS is selective laser melting (SLM), in which very high laser energy is applied to fully melt metals into a solid homogeneous mass. Different CoCrMo alloys meeting the requirements for tissue applications have been investigated to observe the effect of the laser melting process on corrosion and metal release in biologically relevant fluids . The str−3 and acts as a binder for the stainless steel particles. This technique needs an additional heating cycle in which the polymer is burned out and the green part is further sintered and infiltrated with, e.g., bronze to reach a high density. The second technique is used for Ti6Al4V or CoCr alloy and consists of melting the metal powder completely by a laser beam with an energy density of 200 J mm−3, avoiding the need for post-processing. Further surface modification of the laser-melted Ti6Al4V alloy has shown improvement in biocompatibility and a reduction in post-implant complications [Dental implants have been fabricated from stainless steel and Ti6Al4V and CrCo alloys by the SLM method . For theications . The allications .2.1.4.Polymers are elastic and have low stiffness, whereas ceramics are rigid and brittle . By mixiNumerous studies have been done to evaluate the potential of SLS in producing composite scaffolds containing polymer and ceramic , 71, 72.Tg and to bind and partially expose the HA particles within the sintered PEEK matrix.A study of scaffolds consisting of microspherical calcium phosphate (CP)/poly(PHBV) and carbonated hydroxyapatite (CHA)/poly(L-lactic acid) (PLLA) has shown an improvement in biological properties. Laser-sintered HA/polyetheretherketone (PEEK) can also satisfy the requirements of tissues and scaffolds . PEEK, aUp to now, there have been few works regarding the fabrication of porous CS scaffolds using SLS and enhancing their mechanical properties by adding HA whiskers at the same time. In a previous study, porous scaffolds from CP materials with different weight ratios of TCP/HAP  were fabricated via SLS .2.2.et al [1/2 for a scaffold made from high surface area HA nano powder by using SLS with a laser energy density of 4 J mm−2. Shuai et al [et al [Depending on the material and physical properties of the final products, various mechanical properties can be obtained for fabricated scaffolds. A high compressive strength of 18.2 ± 1.2 MPa has been reported for laser sintered CS scaffolds with an interconnected pore structure . Shuai eet al  have repai et al  have fabl [et al  have beeet al [2 carbon atoms, has attracted great interest for producing the next generation of nanocomposites used in scaffold fabrication [1/2 with a graphene content of 0.5 wt%, indicating significant improvement of 105% and 38% respectively compared with pure 58 S.An interesting work done by Gao et al  has presrication . Due to et al [2, 4% P2O5, 20% CaO, 5% MgO, 6% Na2O, and 12% K2O (wt%) for SLS scaffold fabrication. The compressive strength of the fabricated scaffolds was studied for up to two months when immersed in Dulbecco’s modified eagles medium (DMEM). The compressive strength of the parts decreased from 40 ± 10 MPa in the dry condition and 26 ± 6 MPa after 60 days. Porous biocompatible pure Ti and nitinol (NiTi) alloy was also successfully sintered into 3D scaffold form using a Nd:YAG laser with energy input of 100–300 J cm−2 [2 lasers with respect to metallic powders due to better absorbance at shorter wavelengths [Velez et al  have rep0 J cm−2 . Nd:YAG elengths . Applyin2.3.In vitro and in vivo tests play an important role in the biological assessment of biomaterials for the fabrication of tissues and scaffolds [caffolds . A numbeet al [μCT confirm a remodeling of up to ∼400 μm into the microstructure of the struts. Alamar blue and alkaline phosphate activity (ALP) assays revealed that in general, in the initial period composites with lower HA content (15 wt%) showed better metabolic activity compared with those having higher HA content; however, by day 14 the performance of the two compositions was equal [One issue regarding scaffolds made from polymers is the hydrophobic nature of their surface, which results in the negligible availability of bioactive sites . The preet al  have repet al . PCL scaet al . The addet al . The proas equal .et al have fabricated scaffolds from Nylon-6 by SLS [Das 6 by SLS . BiocompIn vitro tests of 3D scaffolds have demonstrated that the incorporation of CP nanoparticles significantly improves cell proliferation [In vitro results have also revealed that the addition of bioactive CS ceramic into a PVA matrix (<20 wt%) enhances the bioactivity of scaffolds, i.e., the number of MG-63 cells attached to the surface of the composites increases in the presence of higher amounts of CS in the scaffolds [feration  and alkacaffolds .et al [in vivo via gene therapy. Histological evaluation and μCT data show that the interior pore architecture of laser sintered PCL scaffolds can induce bone generation in vivo. Lohfeld et al [In vivo, a PCL/TCP composite scaffold showed inferior behavior compared with the reference material (β-TCP) with respect to a critical size defect regarding the promotion of bone regeneration, scaffold degradation, and inflammatory reaction. Saito et al [Williams et al  have shold et al  have proto et al  have exaMore studies regarding the biological properties of laser sintered tissues and scaffolds from different materials are summarized in table 3.The binder jetting process is another AM technique which employs inkjet head (IJH) technology for processing materials. In this system, the head prints a liquid binder onto thin layers of powders based on object profiles that have been generated by software . Two kinWhether to use thermal or piezoelectric inkjet printers depends on the desired properties of the final part. Each inkjet technique has some points which can be categorized as availability, printing speed, accuracy of printed parts, and functional cost. Thermal inkjet printers have some advantages, including availability, higher print speed, and lower cost of parts fabrication compared with piezoelectric inkjet printers . HoweverOn the other hand the advantages of piezoelectric inkjet printers include the capability to generate and control uniform droplet size and ejection directionality as well as to avoid exposure of the binder to heat stressors . The sheAs the precision of fabricated models strongly depends on the velocity, initial size, and path of the droplets, it is essential to control the parameters, including nozzle diameter, binder properties, and resonance frequency of the head, which have a direct and indirect effect on these terms .3.1.In general, a wide range of powders including ceramics and polymers can be processed by inkjet 3DP; however, binder selection is a key factor in successful part fabrication. This section provides a detailed discussion of the existing powders and binders which are used for the fabrication of tissues and scaffolds.3.1.1.−1, respectively. To obtain the aforesaid range, the ratio of Re is the Reynolds number We is the Weber number V and r are droplet velocity and radius respectively [The materials used as a binder must have suitable properties to prevent spreading from nozzles. To adjust the fluid properties of the organic suspensions to be compatible with the type of printing head, the viscosity and surface tension must be 5–20 Pa.s and 35–40 mJ Nectively –107. Wherecision . Three drecision , phosphorecision , and polrecision . DependiAlthough polymeric binders have been widely used to fabricate ceramic parts, the final products suffer low resolution and mechanical strength. Using an acid binder solution has been suggested to improve the resolution and mechanical properties of the printed parts . Lyophil3.1.2.Flowability of powders is an essential parameter for 3DP processing. Sufficient flowability of powders allows the roller to build up thin layers, leading to high 3DP resolution. Too little flowability decreases fabrication resolution due to insufficient recoating. On the other hand, very high flowability does not provide sufficient powder bed stability for 3DP.Wettability of particles is another factor in 3DP processing. The volume of binder distributed into the powder bed and also the amount of binder absorbed by the powders determines the resolution (voxel size) and mechanical properties of the parts. It has been confirmed that too-low wetting of fine powder particles results in powder bed rearrangement that is possibly detrimental to further 3DP, and too-high wetting and slow powder reaction will reduce the smallest feature size –115. TheFor powder materials, a broad range of polymers, ceramics, and composites can be applied in the field of tissue engineering. As has been previously described the binding mechanism of bioceramic powders used in binder jetting systems is based on the hydraulic setting reaction –119. WheCP has been widely applied in inkjet printing , 120. CP3.2.et al [et al [Improving the mechanical properties of porous parts is a challenge in inkjet 3DP. In some cases, to reach a suitable strength, the scaffolds are sintered after printing. This post-processing exposes the final part to failure due to the burnout of binder which is present or because of high binder concentration. Therefore, the binder concentration must be minimized while still providing sufficient mechanical stability to the printed structure. Moreover, sintering causes a dimensional change in the final part . Tarafdeet al  have repl [et al  have fabet al [β-CS scaffold using 12% PVA solution as a binder. The compressive strength and Young’s modulus of printed CS scaffolds with a pore size of 1 × 1 mm and porosity of 65% were 3.6 ± 0.1 MPa and 40 ± 8 MPa. A study of the deformation of scaffolds during the compressive test revealed that the printed CS scaffolds partially maintained a scaffold configuration in the center position and only the border area collapsed. This may be interpreted as the effect of the proper distribution of polymeric binder on the flexibility of the printed scaffolds.As mentioned in section et al  have preBy comparing the compressive strength of CS scaffolds with those using polyurethane (PU) foam and PDLLA solutions to bind the particles, the influential role of binders in inkjet 3DP can be seen. The strengths of scaffolds prepared using PU and PDLLA solutions as binders were 0.3 and 1.45 MPa, respectively, i.e., significantly lower than when using a PVA solution .et al [β-TCP as the powder and phosphoric acid as the binder. They improved the compressive strength by adjusting the volume ratio of binder to powder. Compressive strengths of 3.4 and 7.4 MPa were obtained for a binder-to-powder-volume ratio of 2 and 4, respectively. Another study showed that an 8.75 wt% phosphoric acid solution binder can improve mechanical strength while retaining cell viability at 68% ± 6%. As a surfactant, 0.25 wt% Twin 80 was added to the binder solution to improve printability [In the case of acidic binders, Vorndran et al  have fabtability .4  composites it was confirmed that using very fine HA powders (≤20 μm) leads to a loosely packed powder bed and thus a high level of heterogeneity, which results in slow drop penetration, large drop penetration depth, low wetting ratio, and poor green mass and green strength for the final 3DP components. On the other hand, using coarser HA powders (30–100 μm) can show higher mechanical strength values [X-axis scaffolds has been reported as 0.76 ± 0.02 MPa, whereas this value is 0.88 ± 0.02 MPa along the Y-axis. Despite exhibiting a higher compressive strength, scaffolds printed along the Y-axis have been shown to contain traces of PVA degradation products after heat treatment. Using metal oxide components as a reinforcement agent is also recommended to improve the mechanical properties of bioactive ceramics, especially for hard tissues and implant applications [2/ZnO to TCP can increase the mechanical properties of implants. For investigation of this effect, Fielding et al [2/ZnO. Cylindrical scaffold CAD files were created with interconnected square channels of 1000 μm, 750 μm, and 500 μm sides and 7 mm diameter and 10.5 mm height. The doped fabricated scaffolds, which had less total open pore volume than the pure scaffolds, showed the greatest compressive strength, with the 1000 μm, 750 μm, and 500 μm green channel sizes at 10.21 ± 0.11 MPa, 8.2 ± 0.4 MPa, and 4.34 ± 0.3 MPa, respectively. The pure samples with the green channel sizes 1000 μm, 750 μm, and 500 μm had average compressive strengths of 5.48 ± 0.04 MPa, 2.7 ± 0.2 MPa, and 1.8 ± 0.2 MPa, respectively.As mentioned earlier, the size of the powder particles has a direct influence on the mechanical strength of the parts. In HA/CaSOh values . Printinh values . The mecications , 140. Mong et al  fabricat3.3.Apart from having good mechanical properties, tissues and scaffolds fabricated by inkjet 3DP must be able to react with cells after implantation. Improving the biological properties  of printed tissues depends on the properties of powders and binders, on pore volume, and also on post-processing of printed tissues.et al [As previously discussed, in some cases poor mechanical strength of printed tissues can be improved by the sintering process. However, sintering may also compromise biodegradability due to increases in the crystallinity of printed parts, leading to poor resorption by osteoclasts . The binet al . Althouget al .et al [in vivo tests and cell seeding, it was concluded that 3D-printed hydroxyapatite and 3D-printed TCP as well as bovine HA blocks are biocompatible for cells derived from a human periosteum.A report by Becker et al  has presin vitro and in vivo and also increases the ALP of TCP/zinc oxide composite, which is an enzymatic marker for osteoblastic differentiation [Studies have shown that zinc oxide has a stimulatory influence on fabricated tissue formation ntiation , 143.μm, providing pores of this size or larger is essential for bone grafting. Depending on whether post-processing is used, pores with the desirable geometry can be created by considering the pore size and geometry in the primary design of the structure or can be derived from porogens burned out during sintering. Pore geometry is known to be an important factor in determining bone healing response [+, Mg2+, and Sr2+ have led to excellent improvement in the biological properties of HA, only a few studies have investigated the effect of cation doping on the 3D interconnected porosity of 3D printed tissues and scaffolds. Both micro and interconnected macropores facilitate the infiltration of osteoprogenitor cells, which emphasizes the presence of multiscale porosity in tissue engineering scaffolds. In a research conducted by Tarafder et al [2+and Sr2+ in a TCP structure and their influence on 3D printed bone tissues led to pore sizes of 245 ± 8 μm and 311 ± 6 μm for doped and pure TCP scaffolds, respectively, which were close to the designed pore size of 350 μm [Since bone can grow into pores with a diameter of approximately 300 response . The adder et al , the pref 350 μm . As showin vivo study has also revealed the effect of pores on bone formation after implantation, i.e., cylindrical holes 2 mm in diameter running across 3D printed tailor-made bone implants (TIs) showed that bone formation on a larger scale was facilitated [Another ilitated . Based oilitated .4.AM offers unique advantages with respect to fabrication tissues and scaffolds with a complex external anatomy shape and internal porous structure. Coupling complicated porous 3D design with AM techniques can create a range of bone tissues and scaffolds from various materials. Among the AM techniques, inkjet 3DP and SLS are two powder-based tools which are widely used for biomedical engineering applications.In the case of SLS, the initial setting, e.g., laser power and scanning speed, is crucial. Without any modification, commercial SLS machines can be used only for small amounts of powdered materials for fabricating specific biomedical applications. Different research groups have begun to optimize the SLS parameters for fabricating special objects with a desired 3D porous architecture in minimum fabrication time and at minimal cost. For tissue engineering, control over mechanical behavior while retaining the designed porous structure is very important. This issue can restrict the use of pure biocompatible polymers. Another disadvantage of the SLS technique for scaffold fabrication is that hydrogels cannot be processed, and it is also impossible to encapsulate cells in scaffolds . The lacμm). Trapped powders inside the pores may well sinter with the porous part, making it less interconnected than the designed part. Such problems with loose powders can reduce the dimension of the pores after sintering.In the case of using inkjet 3DP machines for tissue engineering, although this method can be employed for fabricating tissues with defined shapes and porous architecture from almost all ceramics and polymer materials, the selecting of a suitable binder is still a challenge which needs extensive optimization. Among the binders, acidic ones can provide good mechanical properties for fabricated tissues; however, binder residue in printed structures is difficult to remove and may make tissues toxic. In most cases, post-processing such as sintering is required for printed parts to achieve the desired mechanical behavior. During the sintering process, parts shrink, and unfortunately the shrinkage is not necessarily uniform. The first effect of non-uniform shrinkage on sintered parts is cracking, which make the parts useless. Since the outside part of bone is denser than the inner part, mimicking such structures is very difficult using 3DP, which is the second challenge related to non-uniform shrinkage during sintering . AnotherApart from the issues related to SLS and inkjet 3DP settings as well as selecting suitable biomaterials, clinical usage of AM processed parts is still a big challenge. In fact, there are many obstacles along this long and difficult road.The gap between the concept and the clinical use of tissue engineering comprises three main factors: the need for understanding native-tissue characterization, the need to incorporate this characterization into tissue design, and finally, the necessity of fabricating tissues based on these design specifications. Despite all the advances in biomaterials science, there are still major gaps in this field relative to the surface chemistry, growth factor release, and mass-transport characteristics that best accelerate a specific tissue formation. Therefore, there are no strategies specifying which material is appropriate for tissues, which linear or nonlinear elastic properties a scaffold should exhibit, which surface chemistry a scaffold should have, or which permeability or diffusion properties a scaffold should demonstrate. In addition to these gaps and challenges, the clinical use of artificial tissues and scaffolds needs volunteer patients for bone tissue replacement surgeries. Because this field is still new and not much clinical surgery has been done, this high-risk surgery might pose challenges after implantation.in vitro and in vivo experiments, whereas implantations of scaffolds in the human body are still rare. The union between produced parts and host bones is affected by dimensional compatibility, biodegradability, pore size, and pore interconnectivity. Saijo et al [α-TCP powder with 10 μm particle diameter and a mixture of 5% sodium chondroitin sulfate, 12% disodium succinate, and 83% distilled water as a curing solution. The scaffolds showed rapid union in 10 patients at 12 months after implantation, which can be attributed to the implant macropore structure resulting in rapid cell growth [et al [Few SLS and ink jet printing products have been used in clinical applications. Most reports have been limited to using models as guide templates for surgery and for jo et al  have repl growth . Recentlh [et al  reportedDemand for AM technologies such as SLS and 3DP will increase in the future due to their capability to make custom medical devices that can be tailored for patient-specific and defect-specific clinical needs. Integrating all key points mentioned as well as finding solutions to cope with the challenges and issues are important in guiding the progress of these techniques toward achieving the objective of clinical use.


Most conventional treatment for musculoskeletal conditions continue to show moderate effects, prompting calls for ways to increase effectiveness, including drawing from strategies used across other health conditions. Therapeutic alliance refers to the relational processes at play in treatment which can act in combination or independently of specific interventions. Current evidence guiding the use of therapeutic alliance in health care arises largely from psychotherapy and medicine literature. The objective of this review was to map out the available literature on therapeutic alliance conceptual frameworks, themes, measures and determinants in musculoskeletal rehabilitation across physiotherapy and occupational therapy disciplines.A scoping review of the literature published in English since inception to July 2015 was conducted using Medline, EMBASE, PsychINFO, PEDro, SportDISCUS, AMED, OTSeeker, AMED and the grey literature. A key search term strategy was employed using “physiotherapy”, “occupational therapy”, “therapeutic alliance”, and “musculoskeletal” to identify relevant studies. All searches were performed between December 2014 and July 2015 with an updated search on January 2017. Two investigators screened article title, abstract and full text review for articles meeting the inclusion criteria and extracted therapeutic alliance data and details of each study.One hundred and thirty articles met the inclusion criteria including quantitative (33%), qualitative (39%), mixed methods (7%) and reviews and discussions (23%) and most data came from the USA (23%). Randomized trials and systematic reviews were 4.6 and 2.3% respectively. Low back pain condition (22%) and primary care (30.7%) were the most reported condition and setting respectively. One theory, 9 frameworks, 26 models, 8 themes and 42 subthemes of therapeutic alliance were identified. Twenty-six measures were identified; the Working Alliance Inventory (WAI) was the most utilized measure (13%). Most of the therapeutic alliance themes extracted were from patient perspectives. The relationship between adherence and therapeutic alliance was examined by 26 articles of which 57% showed some correlation between therapeutic alliance and adherence. Age moderated the relationship between therapeutic alliance and adherence with younger individuals and an autonomy support environment reporting improved adherence. Prioritized goals, autonomy support and motivation were facilitators of therapeutic alliance.Therapeutic Alliance has been studied in a limited extent in the rehabilitation literature with conflicting frameworks and findings. Potential benefits described for enhancing therapeutic alliance might include better exercise adherence. Several knowledge gaps have been identified with a potential for generating future research priorities for therapeutic alliance in musculoskeletal rehabilitation.The online version of this article (doi:10.1186/s12913-017-2311-3) contains supplementary material, which is available to authorized users. Conventional treatments such as exercise commonly used in the management of musculoskeletal (MSK) conditions continue to show only moderate effects –3. ReseaTA has been studied extensively across a range of psychotherapy treatment modalities and aetiologies , 10 withFurthermore, it remains unclear whether patient or therapist characteristics most determine outcome  and despBased on these shortcomings in the TA literature, a comprehensive review of primary research in TA is required to map the breadth of literature for MSK conditions to advance knowledge in the following areas: conceptualization, active ingredients, psychometrically sound measures, mechanism of effect, and the mediating, moderating or predicting effect of TA on adherence. To this end, we conducted a scoping review of TA in MSK practice informed by the disciplines of OT and PT. The purpose of the study was to describe the type of research conducted to investigate the relationship between TA and rehabilitation of MSK conditions. Specifically, this review intends to describe to what extent the literature has theoretical underpinnings or a common understanding of what constitutes TA, has addressed the relationship between TA and adherence to treatment or treatment outcomes and how TA is measured.This scoping review was informed by Levac et al.  and Arksphysiotherapy or occupational therapy were completed to identify experimental studies that discussed or investigated the relationship between TA and adherence to exercise in the management of adults with MSK conditions. Using a combination of key words and medical subject (MeSH) terms  and selected if they met the following criteria:  quantitaData related to TA were extracted from articles meeting the inclusion criteria by the lead author (FB) and reviewed by a second author (JM). Each article was first categorized based on study methods  and level of care . The following information was extracted and synthesised in summary format from the articles: authorship, publication year, country, setting, discipline, aims, design, participants, themes, and findings. Secondary data extracted included information on the conceptualization of TA; frameworks, theories or models, relationship between TA and adherence to exercise, measures of TA and participant perspectives on TA themes.Descriptive statistics were calculated to summarize the data. Frequencies and percentages were used to describe nominal data . A narrative synthesis approach , 38 facin = 426), titles from nursing (n = 160) and medicine (n = 263) and titles from psychotherapy (n = 430) literature. After final abstract and full text screening, 130 articles were selected as listed in (1 s–130 s)  between 2011 and 2016 and most of the studies originated from the PT discipline as depicted in Fig. Participants included 7,018 patients, 1225 OTs and 994 PTs. By country, most of the publications originated from the USA (23%) or Australia (16.9%). By continent, Europe accounted for 44.6% of the studies as compared to 30% from North America and 23% from Australasia. There was only one study published in South America, only 2 studies from Asia and none from Africa. The earliest study dates to a 1981 with an increase in publications  than qualitative studies  as shown in Table Overall, there were more quantitative Additional file 2:Therapeutic Alliance Terms. Description of therapeutic alliance terms detailed in included studies. (DOCX 63 kb)Additional file 3:Perspectives on therapeutic alliance from participants. This file highlights the perspective and experiences of participants on therapeutic alliance. (DOCX 400 kb)


Aim. This report introduces extrapedicular infiltration anesthesia as an improved method of local anesthesia for unipedicular percutaneous vertebroplasty or percutaneous kyphoplasty. Method. From March 2015 to March 2016, 44 patients  with osteoporotic vertebral compression fractures with a mean age of 71.4 ± 8.8 years (range: 60 to 89) received percutaneous vertebroplasty or percutaneous kyphoplasty. 24 patients were managed with conventional local infiltration anesthesia (CLIA) and 20 patients with both CLIA and extrapedicular infiltration anesthesia (EPIA). Patients evaluated intraoperative pain by means of the visual analogue score and were monitored during the procedure for additional sedative analgesia needs and for adverse nerve root effects. Results. VAS of CLIA + EPIA and CLIA group was 2.5 ± 0.7 and 4.3 ± 1.0, respectively, and there was significant difference (P = 0.001). In CLIA group, 1 patient required additional sedative analgesia, but in CLIA + EPIA group, no patients required that. In the two groups, no adverse nerve root effects were noted. Summary. Extrapedicular infiltration anesthesia provided good local anesthetic effects without significant complications. This method deserves further consideration for use in unipedicular percutaneous vertebroplasty and percutaneous kyphoplasty. Since the first reports of percutaneous vertebroplasty procedures in 1987, percutaneous vertebroplasty (PVP) and percutaneous kyphoplasty (PKP) have been widely used to treat osteoporotic vertebral compression fractures, myeloma, and vertebral metastases. Advantages of the procedure include minimal surgical invasiveness, good pain relief, and rapid recovery .Recent reports have claimed similar biomechanical stability and clinical efficacy of unipedicular PVP (PKP) as compared to bipedicular PVP (PKP). Therefore, the unipedicular procedure is advocated by most surgeons –6. LaterBecause of this, it is important to have adequate anesthesia in the extrapedicular region. Accordingly, the authors have added extrapedicular infiltration anesthesia (EPIA) to the conventional local infiltration anesthesia (CLIA) and report the retrospective study of comparing anesthetic effect of CLIA + EPIA with CLIA as a control group for unipedicular PVP (PKP).From March 2015 to March 2016, 44 patients  with osteoporotic vertebral compression fractures with a mean age of 71.4 ± 8.8 years (range: 60 to 89) received PVP (PKP). According to the local anesthetic methods, all patients were divided into two groups: 20 patients were managed with CLIA + EPIA and 24 patients with CLIA. Patient perceptions of intraoperative pain were measured with the visual analogue score (VAS). Each patient had brief preoperative teaching to report VAS of 0 as no pain and 10 as maximal pain and to know that they would be asked to report their pain using this scale. Patients were also told that, in the event of severe pain, they could receive additional sedative analgesia. From the time that anesthesia was established to the time that the surgery was completed, patients were asked to report any adverse nerve root effects such as hypesthesia or numbness and any reduction of lower limbs muscle strength. The retrospective study was approved by the ethics committee of the unit and patients provided consent for treatment.The anesthetic process of CLIA group included two steps. For the first step, the projection point of the pedicle was located, and a number 7 epidural needle  was used for anesthesia; from a point 1 cm lateral to the pedicle projection point, 5 mL of 1% Lidocaine Hydrochloride was used to infiltrate the skin, subcutaneous tissue, and part of the lumbodorsal muscles. In the second step, the anesthetic needle was angled 10–15° with the sagittal plane and was directed toward the laminar periosteum at the pedicular projection point on the lamina where 5 mL of 1% Lidocaine Hydrochloride was injected. Besides the two steps, the anesthetic process of CLIA + EPIA group additionally included the third step, called EPIA. For this step, the anesthetic needle tip first was withdrawn to the subcutaneous tissue. It was then directed toward the lateral half of the pedicle along the lateral superior articular process and the superior border of the transverse process , and with no withdrawal of blood, 5 mL of 1% Lidocaine Hydrochloride was injected . The tot t-test, with the level of significance set at P < 0.05, using SPSS version 19.The age, BMI, and VAS between groups were compared using the unpairedP > 0.05). VAS of CLIA + EPIA group was 2.5 ± 0.7 (range: 1~3), while for the CLIA group, it was 4.3 ± 1.0 (range: 3~7), and there was significant difference (P = 0.001) between the two groups. In CLIA + EPIA group, no patients required additional sedative analgesia, but in CLIA group, 1 patient's VAS was 7 and additional sedative analgesia was required. In the two groups, no patients reported nerve root adverse effects.Patients' demographic characteristics and the results in the two groups were shown in The anesthetic method for PVP (PKP) usually includes local anesthesia, sedative analgesia, and general anesthesia. General anesthesia has been proven to be effective for surgical procedures and is amenable to intraoperative monitoring and management. However, elderly patients undergoing PVP (PKP) may have poor tolerance of general anesthesia because of frequent concomitant circulatory, respiratory, nervous, and endocrine disorders. Moreover, endotracheal intubation and general anesthesia easily cause respiratory complications, and intraoperative neurological symptoms may not be discovered in a timely manner. Additionally, sedative analgesia with opioids and benzodiazepines carries the potential for respiratory depression, called opioid-induced respiratory depression (OIRD) . ClinicaBiomechanical and clinical studies have reported that vertebral axial compressive strength would be largely recovered if bone cement is limited to hemivertebra and that bilateral vertebral rigidity would be obtained if bone cement extended past midline . UnipediUsually, local anesthetic for PVP (PKP) ranges only from the skin to the laminar periosteum, essentially only including the first and the second steps of anesthesia. Because of the laminar barrier, the anesthetic is directed mainly toward the outer surfaces of the lamina and paraspinal muscles, and the extrapedicular periosteum and deeper extrapedicular soft tissue are not anesthetized. However, the acute angulation of the puncture needle will result in penetration of the extrapedicular region, which may result in severe pain. EPIA can anesthetize the entire extrapedicular periosteum and soft tissues by directing the anesthetic needle to the lateral 1/2 of the pedicle and infusing local anesthetic. To avoid nerve root anesthesia and lumbar artery damage, the needle should not enter too deeply into the junction of pedicle and vertebral body. C-arm X-ray monitoring can aid with precise localization of the anesthetic needle. In the event of articular process hyperplasia at lower lumbar levels, angulation of the anesthetic needle could be increased to 10–15°. At thoracic vertebral levels, it is safe to contact the ribs, but care should be taken to avoid piercing the pleura.EPIA deserves clinical consideration for unipedicular PVP (PKP), as it provides effective anesthesia and presents no significant complications. However, it may slightly increase X-ray radiation exposure for precise localization of the anesthetic needle.


Neoadjuvant chemotherapy (NAC), the systematic chemotherapy given to patients with locally advanced and inoperable breast caner, has been proven to be of great clinical values. Many scientific reports confirmed NAC could effectively eliminate sub-clinical disseminated lesions of tumor, and improve long-term and disease-free survival rate of patients with locally advanced breast cancer (LABC); however, up to now, LABC is still a serious clinical issue given improved screening and early diagnosis. This study, with main focus on inoperable LABC, investigated the values of NAC in converting inoperable LABC into operable status and assessed the prognosis. Sixty-one patients with inoperable LABC were initially treated with neoadjuvant chemotherapy; their local conditions were improved to operable status. Radical surgery was exerted on 49 patients. Original chemotherapy was performed after surgery, followed by local radiotherapy. And endocrine therapy was optional according to the hormone receptor status. The quality of life for most patients with skin diabrosis was obviously improved because their local conditions were under control. For all recruited cases, the survival duration and life quality were significantly improved in patients who finished both NAC and surgery compared to those who did not. Further more, this study demonstrates improved prognostic consequences. Neoadjuvant therapy is the standard care for treatment of locally advanced and inoperable breast cancer. Application of this systematic therapy before surgery benefits patients with improved rates of breast-conserving surgery, increased possibility of early measurement of response, as well as potentially preferable outcomes for certain subgroups of high-risk patients. Although there is an exponential increase in the number of patients with earlier-stage operable breast cancers in the setting of improved screening and early diagnosis, locally advanced breast cancer (LABC) is still a severe clinical issue. Currently the definition of LABC is a widely used one without standard guidelines due to variations in LABC populations. Usually it is characterized by the following features: the diameter of primary lesion larger than 5 cm T3), adhered and fixed skin to the chest wall T4), fused regional axillary nodes N2), and excluding remote metastasis1, and exc, adhered, fused r5LABC can be classified into two types: operable and inoperable LABC. For the operable LABC, the typical therapeutic scheme is radical surgery combined with additional chemotherapy and radiotherapy. However, simply surgical resection may not be sufficient to excise the whole lesion in a case of chest wall adherence complicated with skin ulcer and edema. Previous attempts to treat such lesions were mostly characterized by early local recurrence and distance metastases, thus these cases were categorized into inoperable LABC79Therefore, the aim of the study was to assess the effectiveness of NAC for patients with inoperable LABC and to further evaluate the prognosis of the patients who could be potential beneficiaries of radical surgery and synthetic therapies due to the effectiveness of NAC.A total of 1705 patients diagnosed with breast cancer were treated in our hospital from March 2004 to May 2009, among which 61 cases had primary inoperable LABC. The characteristics of these inoperable LABC included huge size tumors, ulceration, wide invasion or large inflammatory breast cancer, and no evidence of metastases. All patients were female, with ages ranging between 31 and 71 years old (average age: 55.1 years old). Pathology of theses cases indicated that 50 cases were infiltrating duct carcinoma, 5 cases of infiltrating lobular carcinoma, 2 cases of mucinous adenocarcinoma, 1 cases of squamous carcinoma, and 3 cases of medullary carcinoma. Finally, total 61 patients with primary inoperable LABC were enrolled for the present study.2+ Epirubicin 90 mg/m2+ Cyclophosphamide 600 mg/m2) or TE (Paclitaxel 175 mg/m2 + Epirubicin 90 mg/m2). Prior to the third cycle of chemotherapy and surgery, US was utilized again to evaluate the size of tumor and LN of the affected breast. After completion of chemotherapy, MRI was performed once again to assess any residual disease. And what’s more, appropriate surgical treatments were applied to patients respectively according to their extents of residual tumor, followed by scheduled adjuvant radiotherapy. Due to poor therapeutic effect or some other reasons, there were 12 patients gave up the treatments after 1–4 cycles of NAC.First of all, in order to obtain baseline characteristics of each patient, a complete physical examination and an interview were required to take by all the 61 patients. Clinical examination, mammography (MMG), ultrasound (US), and magnetic resonance imaging (MRI) were applied at the time of initial diagnosis to assess the status of primary tumor in the breast and lymph node. In addition, physical and special examinations were performed to exclude distant metastasis, such as bone scans and alkaline phosphatase detection were performed for detecting bone metastasis, chest X-ray and/or CT for examining lung transfer, liver ultrasound for liver metastasis and MRI for brain. After these most common organs were examined and excluded for distant metastasis, LABC was thus defined. If the patients situations were indicated, PET with 18-fluorodeoxyglucose (FDG-PET) or PET combined with computed tomography were performed prior to surgery. The diagnosis of primary breast cancer was established by core needle biopsies, and lymph node metastasis was confirmed by core needle biopsy or fine-needle aspiration or regional ablation at the ulcerated site. Then systemic chemotherapeutic tolerance of each patient was evaluated including blood cell count, liver function, ECG, chest X-ray or/and CT, bone scan, alkaline phosphatase detection and liver ultrasound. Of all the 61 cases, 49 cases acquired regional improvement after receiving 4 to 6 cycles of NAC and met the standards for radical surgery. The chemotherapeutic schemes included the methods of FEC (5-FU 600 mg/mwww.sgpath.ch); in particular, with respect to the hormone receptors, the percentage of neoplastic cells involving in expression of either ER or PR was recorded, whereas Her2 expression was judged according to the guidelines of approved HercepTest by the Food and Drug Administration. Results were included in the histopathologic diagnostic report and communicated to the clinicians, as well as recorded in the dataset. Breast cancers with less than 5% of ER and PR expressions in neoplastic cells were considered as negative for hormone receptor expression. Weak or incomplete membrane staining (score 0 or 1+) was considered as Her2 negative, whereas strong and complete membrane staining (score 3+) indicated Her2 positive. There wasn’t any cancer with ambiguous expression of Her2 (score 2+) in the studied cases, all the them were either Her2 negative or Her2 positive. In this study, simple classification based on the expressions of ER, PR and Her2 was opted and advantageous since these three markers are routinely carried out in pathology laboratories, staining and evaluation protocols are well established worldwide and quality control programs are already available in several countries. We therefore classified breast cancer cases into four subtypes: luminal A ; luminal B ; BCL  and Her2/neu . Our clinical trial was successfully registered in the WHO International Clinical Trial Registry on May 29, 2014 with registration number ChiCTR-TRC-14005019. It was also approved by the Ethical committee of Southwest Hospital of the Third Military Medical University for carrying out this study with all the patients providing written informed consent. All experiments for this study were performed in accordance with relevant guidelines and regulations.IHC staining for ER , PR  and Her2/neu  were carried out on formalin-fixed, paraffin-embedded tumor samples, prospectively in the context of the routine diagnostic service, using an automated staining system . Tissue sample expressing the antigen of interest were included on each histologic slide for positive control. IHC staining was evaluated by the local pathologists in accordance with national guidelines  or progestin receptor (PR) was positive with Tamoxifen for premenopausal women and aromatase inhibitors for menopausal women. Other chemotherapeutic schemes were chosen if the recurrence or metastases occurred.Survival rate analysis was performed using standard statistical computer software SPSS for windows (Version 13.0). Chi-squared test was used to determine the difference between groups, and the difference was considered statistically significant if P value < 0.05.The study was approved by the Ethical committee of Southwest Hospital of The Third Military Medical University and Institute and written informed consent was provided by the patients. All experiments were performed in accordance with relevant guidelines and regulations.The baseline clinical characteristics of the study population are shown in Post operation follow-ups were conducted at least once every half year with follow up periods ranging between 1 to 6 years. There were 8 cases were followed up for more than 5 years, 19 cases more than 4 years, 29 cases more than 3 years, 42 cases more than 2 years and 49 cases more than 1 year. Eighteen cases suffered local recurrence on the skin of chest wall, inner mammary region or supraclavicular lymph nodes. Recurrence occurred in 4 cases within 1 year, 12 cases between 1 to 3 years, 1 case between 3 to 5 years, and 1 case after 5 years .Survival statistics of the patients who underwent surgery are shown in After 1 to 6 years’ follow-up, 10 out of the 12 patients who did not undergo surgery died within 1 year and the other 2 patients died in the subsequent year. Among the 49 patients who received surgery, 32 cases are still living, 2 cases survived longer than 5 years (2/8), and 13 cases survived longer than 3 years (13/29) . For theObviously, patients with inoperable LABC acquired satisfactory improvements of local status and became eligible candidates for radical surgery through NAC. After surgical excisions of local tumors, ulcer and unbearable smell complications were eliminated although local recurrence rate and distance metastases rate were still relatively high. However, the patients could eventually resume their normal life with drastically improved living qualities compared with their living qualities before surgery. What’s more, ulceration relapsed in only one case before death of all the cases followed.LABC principally represents primary breast cancer, it is classified into stages of T3, T4 and TXN2, excluding distant metastases and accounting for about 10 to 15% of all newly diagnosed breast cancers12Most clinical researches tailored Anthracycline as the principal agent of NAC scheme, and appended Taxane to improve the therapeutic effect of the chemotherapy1416It is showed from our findings that effective NAC could provide patients of inoperable LABC with operation opportunities. As discussed before, the local recurrence rate within 1 year was 8.16% 4/49), and 55.2% (16/29) within 3 years. What’s more, it shows the trend of declination in 3 years and peaks between 1 to 3 years. The low recurrence rate within 1 year might be attributed to the reasons such as follow-up effects of NAC, adjuvant radiotherapy and endocrine therapy after surgery. After 3 years, most patients died due to distant metastases, which might account for the decreased local recurrence. Comparing with local recurrence rate of LABC, it was 48% within 5 years as reported in previous studies/49, and It is known that the most common parts for distant metastases of LABC including bone, liver, lung, brain, contralateral breast region and local lymph. As for inoperable LABC, distant metastases most often than not may occur without timely intervention although it is usually absent during clinical examinationsIn addition, inoperable LABC is featured with more severe symptoms and advanced classification compared with operable LABC, and subsequently causing higher mortality. The mortality rate after surgery in this study was 4.08% (2/49) in 1 year, 48.2% (14/29) in 3 years, and 75% (6/8) in 5 years. Although the mortality rate after surgery was higher than the total breast cancer population, it was significantly lower than the rates of patients without surgery, which are about 83.3% (10/12) in 1 year and 100% in 2 years. The results indicated that NAC combined with surgery could improve the survivals of inoperable LABC.At last, the life quality of the inoperable LABC patients was dramatically improved after the treatments of effective NAC and surgery. Most patients had lived in great miseries and pain due to the complications of LABC, which includes foul ulcer, regional pains and obvious limb swelling. Therefore it is promising to note that severe ulcers for most cases were healed with no recurrence after effective NAC and surgery, and ulceration relapsed in only one case. And most patients with local recurrence were simultaneously accompanied with distant metastases in some critical organs, becoming the main lethal factors in LABC, thus the regional symptoms usually had not become worse before death. The patients couldn’t benefit with prolonged survival duration, fortunately the qualities of their daily life could be improved by appropriately controll of local symptoms.In summary, inoperable LABC can be improved by NAC converting it into operable status. Although the long-term survival rate of these patients didn’t show obvious increase after systematic and synthetic treatment, the mean survival time and quality of life were considerably improved. As this study is by no mean perfect with short research time duration, limited number of follow-up cases longer than 5 years and incomplete clinical data, however, our findings proved that effective NAC could convert inoperable LABC into operable states, and significantly improve the prognosis. With better future follow-ups and a more comprehensive clinical data, the results of the synthetic treatment of inoperable LABC would be more convincing.Nevertheless, some limitations worth mentioning in this study are the facts that only 61 cases were included and short follow up time varying between 1 and 6 years. The results would be more persuasive if we could include more cases and increase the follow-up time. Still, LABC is a major clinical problem in China and a common presentation in many parts of the world. In this study, it is proved that inoperable LABC can be converted into operable states by effective NAC, and prognosis be improved as well. Despite all of these defects, this article provides strong clinical evidence for LABC treatment.The present study demonstrates that effective NAC can convert inoperable LABC into operable states, and further improve the prognosis.How to cite this article: Wang, M. et al. Neoadjuvant Chemotherapy Creates Surgery Opportunities For Inoperable Locally Advanced Breast Cancer. Sci. Rep.7, 44673; doi: 10.1038/srep44673 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


ZnO nanoparticles (NPs) and nanofibers (NFs) were synthesized by a modified sol-gel method in supercritical conditions and electrospinning technique, respectively. CO and NO2 sensing tests have demonstrated that the annealing temperature and shape of zinc oxide nanomaterials are the key factors in modulating the electrical and sensing properties. Specifically, ZnO NPs annealed at high temperature (700 °C) have been found sensitive to CO, while they displayed negligible response to NO2. The opposite behavior has been registered for the one-dimensional ZnO NFs annealed at medium temperature (400 °C). Due to their adaptable sensitivity/selectivity characteristics, the developed sensors show promising applications in dual air quality control systems for closed ambient such as automotive cabin, parking garage and tunnels.ZnO nanomaterials with different shapes were synthesized, characterized and tested in the selective monitoring of low concentration of CO and NO This interest is mainly due to problems related to pollution and strict regulations for safety taken by several countries in many industrial sectors. Metal-oxide semiconductor gas sensors have attracted great attention due to their advantageous features, such as high sensitivity under ambient conditions, low power consumption and simplicity in fabrication. Although metal oxides have been demonstrated to be sensitive to many gases, e.g., Oer gases ,2.x. Zinc oxide is an n-type direct band semiconductor with a wide band gap (3.3 eV) and find wide ranging applications in varistors [etc. Its conductivity can be tailored by controlling the deviation from stoichiometry and by doping [Among them, ZnO has been shown to be useful material for monitoring various pollutant gases like CO, benzene, NOband gap .3 eV andaristors , transpaaristors , solar caristors , blue/UVaristors , gas senaristors ,9, etc. y doping . For gasy doping .Recent reports show that changes in particle morphology (shape) can dramatically alter electrical characteristics and reactivity of ZnO when interacting with gaseous species . The lin2 and CO in air. The monitoring of CO and NO2 is of utmost importance in the environmental control. CO is a gas produced during incomplete combustion and it is toxic at very low concentrations. NO2 is another environmental pollutant arising from combustion facilities. In confined environments, such as automotive cabin, garage parking or tunnels, high concentrations of these pollutants can create serious hazard for the health and should be continuously monitored and controlled [2 are in the sub-ppm range [In this study, ZnO nanomaterials with different shapes were synthesized, characterized and tested in the monitoring of low concentration of NOntrolled . The conpm range .2 sensing material for the detection of NOx with minimal CO interference [2 can affect the efficiency of such filters, on one hand in relation to a problem of short-circuit for high thickness, and on the other hand under gases with complicated mechanisms as the redox reaction with CO on platinum. To overcome these limitations, more selective sensors which respond to CO and NO2 without any interference of the other, are necessary.Our final aim is to develop sensors to be used in devices focused to detection of these two pollutants for air quality monitoring (AQM) applications. In this regard, there are currently two types of sensing technology available: electrochemical and solid state sensors. The most accurate devices use an electrochemical element . These srference . Neverth2 by means of ZnO-based sensors, is here reported. Even if the morphology of sensing material plays an important role in the gas sensor performance, data in literature on this subject are scarce. Gurlo reviewed, in an recent paper, several case studies related to more important sensing materials such as SnO2, ZnO, In2O3 and WO3 [i.e., over size and shape of faces in a crystal, is required for the development of better sensors with increased selectivity and sensitivity. Therefore, the first step toward this morphological control of the gas sensing properties is the design and synthesis of well-defined nanomaterials which are uniform in size, shape and surface structure. For example, Micro-Chemical Systems manufactures dual element sensors for detecting both reducing gases such as CO and HC’s and oxidizing gases such as NO2 and O3 suitable for air quality monitoring, by designing a sensor sensing layer using proprietary nanostructured metal oxides with special features [A preliminary work, devoted to demonstrate the possibility to detect selectively CO and NO and WO3 . He concfeatures .2 were studied.In this concern, results obtained with solid state sensors based on ZnO nanomaterials synthesized by sol-gel and electrospinning and having different morphology, are here reported and discussed. The effects of the morphology and annealing temperature on the sensing performances to CO and NOZnO nanomaterials were obtained by two different synthesis procedures namely the sol-gel and electrospinning methods. Sol-gel technique has been largely used to synthesize metal oxides for sensing applications. Here, ZnO nanopowders have been obtained by a new approach based on slow hydrolysis of the precursor using an esterification reaction in the alcoholic medium, followed by a supercritical drying in ethanol . Thus, p2, MoO3, SnO2 and WO3, for gas sensors. Electrospun metal oxide fibers are reported to be able in detecting low concentration of gaseous substances with high sensitivity [Electrospinning technique also offers numerous advantages for obtaining metal oxide fibers, such as TiOsitivity . The oneD, deduced from XRD spectrum  and B is the full width at half maximum (FWHM) of the XRD peak. The spectrum shows only diffraction peaks of ZnO, without any additional diffraction peaks. The indexed peaks are related to the hexagonal wurtzite structure according to JCPDS Data Base [To compare the morphological and microstructural characteristics of samples synthesized, Scanning and Transmission Electron Microscopy  and X-ray Diffraction (XRD) analysis have been performed. trum see b and calata Base .SEM images showing the morphology of the NPs and NFs after annealing at 400 °C are reported in Sensing tests were carried out by means of the home-made sensing probe shown in 2, ZnO, and In2O3. The oxidation reaction at the semiconductor surface between the reducing gas and the negatively charged surface-adsorbed oxygen (O− or O2−) is at the basis of this mechanism. This process leads to the production of free electrons as follows:In order to simplify the functioning of the dual sensor device, the single sensors would operate at the same temperature. Preliminary work has been then devoted to find the optimal operation temperature. The temperature of 350 °C has been chosen on the basis of the better performance obtained in terms of sensitivity, selectivity and fast response/recovery time. The injection of produced electrons from the surface to the bulk of semiconducting layer induces consequently a decrease of the sensor resistance proportional to CO concentration. From the obtained results, it appears that the annealing temperature influence the sensor response towards CO. The ZnO NPs sample annealed at 700 °C shows greater response than the annealed at 400 °C one. XRD analysis showed that the average particle size increase with the annealing temperature, from 25 nm for the as prepared ZnO NPs up to about 66 nm for the sample annealed at 700 °C, leading consequently to a decrease of surface area. Then, it can be deduced that the increase of sensitivity observed for the ZnO NPs sample annealed at the higher temperature tested should be attributed to other factors than the increase of surface area. It is well known that the crystallinity systematically increase with the increase of annealing temperature, as well as the formation of a greater number of oxygen vacancies . These f2 is unaffected. Indeed, with NO2 as target gas at concentrations in the sub-ppm level (≤1 ppm), on ZnO NPs samples annealed at different temperatures, the resistance variations are barely discernible from the baseline. This is clearly observed in the calibration curves, registered at the operating temperature of 350 °C for both CO and NO2 gases  and no remarkable degradation of the performances has been noted, indicating the good thermo-mechanical and electrical stability of the sensing layer. Therefore, even if the sensitivity to NOterature , the eas3COO)2 2H2O, in poly  (PVA) (Mw = 72.000) as precursors. The main electrospinning process parameters are: the flow rate of 0.1 mL h−1 and 8 kV electric potential between the needle and the substrate. By carefully controlling the process parameters of electrospinning, a well reproducible morphology of the ZnO NFs during different depositions can be obtained. The collected nanofibers were annealed in air at 400 °C to obtain pure ZnO NFs.ZnO nanofibers (NFs) were synthesized by electrospinning technique using a suspension of zinc acetate, Zn, according to protocol reported in [ZnO nanopowders were prepared by a sol-gel route using zinc acetate as a precursor in methanol. After 15 min under magnetic stirring, the solution was placed in an autoclave and dried in supercritical conditions in presence of ethyl alcohol (orted in . In secoKα1 wavelength of 1.5405 Å. The surface morphology of the films was monitored by means of scanning electron microscopy (SEM) tests carried out with a JEOL 5600LV electron microscope . TEM images were recorded on a Technai G20-Stwin transmission electron microscope  using an accelerating voltage of 200 kV.The microstructure of the samples was investigated by XRD  using the Cu 2 target gas was varied from 0.25 to 2 ppm. A multimeter data acquisition unit Agilent 34970A  was used for this purpose, while a dual-channel power supplier instrument Agilent E3632A was employed to bias the built-in heater of the sensor to perform measurements at super-ambient temperatures. The gas response, S, is defined as S = R0/R for CO and S = R/R0 for NO2, where R0 is the baseline resistance in dry synthetic air (20% O2 in nitrogen) and R is the electrical resistance of the sensor at different CO or NO2 concentrations, respectively, in dry synthetic air.NPs based sensors were made by printing films (1–10 μm thick) of the nano-powders dispersed in water on alumina substrates (6 mm × 3 mm) with Pt interdigitated electrodes and Pt heater located on the backside. NFs based sensors were made by direct spinning of fibers on alumina substrates. The sensors were then introduced in a stainless steel test chamber for the sensing tests. Electrical measurements were carried out in the temperature range from room temperature to 400 °C, under a synthetic dry air total stream of 100 sccm, collecting the sensors resistance data in the four point mode. Gases coming from certified bottles can be further diluted in air at a given concentration by mass flow controllers. The concentration of CO target gas was varied from 5 to 50 ppm, and the concentration of NO2 were measured at working temperatures ranging from 200 to 400 °C. The effects of the annealing temperature and shape on the sensing performances were demonstrated. Sensitive and selective CO sensors based on ZnO NPs annealed at high temperature have been developed, whereas sensitive NO2 gas sensors are based on the ZnO NFs synthesized by electrospinning. The developed sensors show promising applications in dual air quality control systems for ambient air and closed ambient conditions, such as automotive cabin, parking garage and tunnels.In this work, ZnO nanostructures with different morphologies were synthesized and characterized. Their morphologies and microstructures were determined by SEM, TEM and XRD. ZnO NPs with an average grain size of about 25 nm resulted from the sol-gel synthesis in supercritical conditions. By electrospinning method, ZnO NFs were instead obtained. The response of the synthesized nanomaterials to low concentrations of CO and NO


Aquaculture wastewater is one of the most important alternative water resources in arid regions where scarcity of fresh water is common. Irrigation with this kind of water may affect soil microbial functional diversity and community structure as changes of soil environment would be significant. Here, we conducted a field sampling to investigate these effects using Biolog and metagenomic methods. The results demonstrated that irrigation with aquaculture wastewater could dramatically reduce soil microbial functional diversity. The values of diversity indices and sole carbon source utilization were all significantly decreased. Increased soil salinity, especially Cl concentration, appeared primarily associated with the decreases. Differently, higher bacterial community diversity was obtained in aquaculture wastewater irrigated soils. More abundant phyla Actinobacteria, Chloroflexi, Acidobacteria, Gemmatimonadetes and fewer members of Proteobacteria, Bacteroidetes and Planctomycetes were found in this kind of soils. Changes in the concentration of soil Cl mainly accounted for the shifts of bacterial community composition. This research can improve our understanding of how aquaculture wastewater irrigation changes soil microbial process and as a result, be useful to manage soil and wastewater resources in arid regions. This is especially important in arid and semiarid regions where commonly scarcity of fresh water forces farmers to use water from other sources to cultivate thirsty crops3. Wastewater is often proposed as one of these most important water resources. Irrigation with this kind of water could not only significantly relieve pressure on fresh water resources, but also alleviate the discharge of effluents into water environment, avoiding in this way the deterioration of fresh water ecosystems associated with eutrophication and algal bloom4. The use of wastewater in irrigation has also been found to have additional agronomic benefits associated with soil structure and fertility. According to Kiziloglu et al.5, wastewater has a high nutritive value that may reduce fertilizer application rates and increase productivity of poor fertility soils. Diverse studies have indeed shown that wastewater irrigation increases soil organic matter7. However, potential risk follows as a shadow aspect. Detrimental effects of wastewater irrigation on soil quality, such as increase salinity and decrease soil pH9, as well as increase soil heavy metal contamination12, have been reported as a research topic for decades. Meanwhile, irrigation with wastewater is also associated with several biological risks, i.e., the presence of pathogens, viruses, helminthes and protozoa in soils15, which have opened a new controversial front in the public debate16.As the world needs about 60% more food to feed the 9 billion people in 205017. They are not only the principal participants and drivers of biogeochemical cycling of elements18, but also the sensitive indicators of soil environmental change19. Hidri et al.20 found that long-term irrigation with treated wastewater resulted in increased soil microbial abundance and induced in particular compositions of the bacterial and fungal communities. Oved et al.21 and Ndour et al.22 investigated that wastewater irrigation produced shifts in ammonia-oxidizing bacteria population in soils, as compared to soils irrigated with freshwater. However, no difference was found by Ndour et al.22 between the two treatments in microbial biomass or microbial activities (measured as fluorescein diacetate activity). Truu et al.23 reported that it was the willow growth rather than wastewater irrigation affected soil microbiological and biochemical properties under short term municipal wastewater irrigation. As described by Lopes et al.24, the direct microbiological risks associated with the use of wastewater in agricultural irrigation were mainly the disturbance of the indigenous microbial communities in soils and the influence on their functional activities. Nevertheless, our understanding of these aspects has much room for improving.Soil microbes are one of the most complex components of soil ecosystem25. Several studies have focused on the effects of aquaculture wastewater irrigation on soil chemical and physical processes27. To the best of our knowledge, however, up to now few studies assess the comprehensive effects on soil microbial functional characteristics and bacterial community composition due to long-term use of aquaculture wastewater irrigation. In addition, as the next generation sequencing methods have commonly used for mapping soil microbial phylogeny28, it is surprising that little research about the effects of aquaculture wastewater irrigation on soil microbes is involving in this. With this review, we conducted samplings in fresh and aquaculture wastewater irrigated grape fields in a typical arid region to investigate the differences of microbial functional diversity and bacterial community structures in two soils and analyze the relationships of microbial characteristics and the abiotic environment. The main objective of this study is to find out the effects of aquaculture wastewater irrigation on soil microbial properties and analyze the possible reasons. We hope this research can contribute to generate the progress in the understanding of how aquaculture wastewater irrigation changes the structure and function of soil ecosystem by influencing soil microbial process and as a result, be useful to manage soil and wastewater resources in arid regions.Generally, the compound and concentration of aquaculture wastewater are different from municipal and industrial wastewater, which are expected to stimulate different organisms and metabolic pathwaysP < 0.05; Fig. P < 0.05). Under this circumstance, although totally 24 sole carbon sources could be used in AWS, mainly 7 sources had higher values, i.e. cellobiose, glucosamine, glucoside, d-lactose, mannitol, asparagine and tween 80, which accounted for 9.3, 7.5, 6.8, 10.5, 21.8, 7.7 and 11.5% of total substrates consumption respectively.Remarkable differences in microbial functional characteristics were detected in fresh (FWS) and aquaculture wastewater irrigated soils (AWS). The values of average well color development (AWCD), which represented the metabolic activities of soil microbial communities in using carbon sources, were significantly lower in AWS versus FWS  was confirmed. All six carbon categories consumed by soil microbes showed strong positive correlations with the Axis 1, demonstrating that they were together determined the array of the samples.Principal component analysis (PCA) showed that the samples 1, 2 and 3 in FWS (with green color) and 4, 5 and 6 in AWS (with blue color) were clearly separated along the Axis 1 Fig. . MeanwhiP < 0.05, Table P < 0.01). Figure P < 0.01), with classes Actinobacteria and Thermoleophilia in phylum of Actinobacteria increased from 10.6 to 12.0% and 4.3 to 7.4% respectively. Some other classes, albeit the relative abundances were not dramatic, such as Phycisphaerae, Chloracidobacteria and ZB2, were also found richer in AWS.Aquaculture wastewater irrigation could also significantly change the bacterial community structures. The number of bacterial operational taxonomic units (OTUs) and the values of community diversity indices , Shannon and Simpson) in AWS were all significantly higher than in FWS . Phyla OD1, Acidobacteria, Verrucomicrobia, Actinobacteria and Gemmatimonadetes which were more dominant in AWS  were less varied than in AWS (with blue color on the right) as the three samples in AWS were more scattered Fig. . Along tAWS Fig.  showed s4 were significantly and negatively correlated with all carbon substrates' utilization (P < 0.01). Meanwhile, the most difficultly consumed carbon sources, amines and phenols, in AWS were negatively affected by soil Na, Ca and Mg concentrations, demonstrating that these salt ions played an important role in the consumption of these carbon sources. The Axis 1 of redundancy analysis (RDA) showed a clear environmental gradient of increasing P and decreasing K, Cl and SO4, which separated all carbon substrates utilization to the positive direction of the Axis 1 .Soil environment changes which induced by aquaculture wastewater irrigation could significantly influence microbial functional characteristics and community structures. High concentration of soil total phosphorus (P) favored the increase in the consumption of Carbohydrates, Carboxylic acids, Amines, Phenols and Polymers Table . The cons 1 Fig. . Althoug4 concentrations while the abundance of OD1 was negatively correlated with soil P but positively with soil K, Na, Ca, Mg, Cl and SO4 concentrations. Phylum Proteobacteria was mainly affected by soil EC  although no exactly ions were involved and soil Ca concentration was negatively correlated with the abundance of Planctomycetes. RDA showed total 88.4% of the variation in bacterial community structures was explained  analysis. As for the bacterial community composition, the relative abundances of Proteobacteria, Bacteroidetes and Planctomycetes reduced significantly from FWS to AWS while for that of Actinobacteria, Chloroflexi, Acidobacteria and Gemmatimonadetes increased. Generally, the Proteobacteria encompass an enormous level of morphological, physiological and metabolic diversity, and are of great importance to global carbon cycling36. The majority of Proteobacteria are thought to grow fast, preferring nutrient-rich environments and are shown to be positively correlated with soil CO2 production37. Indeed, the observed reduction was consistent with decreased organic carbon in aquaculture wastewater irrigated soil (from 1.38 in FWS to 1.25 g kg−1 in AWS). One possible reason for this result is a significant soil salinity increase under aquaculture wastewater irrigation38. Wu et al.39 showed that the relative abundance of the Betaproteobacteria decreased with increasing salinity, whereas that of the Alphaproteobacteria and the Gammaproteobacteria increased. However, in study of soils from the former lake Texcoco, Valenzuela-Encinas et al.40 showed that the dominant class of Proteobacteria in both high and low saline soils was Gammaproteobacteria, whereas in medium saline soils was Alphaproteobacteria. Different region and soil characteristics may contribute to these differences. In our study, although the abundances of both these three classes decreased in AWS, Betaproteobacteria and Gammaproteobacteria were the significant derivers for the Proteobacteria relative abundance reduction. The phyla Actinobacteria, Chloroflexi, Acidobacteria and Gemmatimonadetes were found the most abundant bacterial groups thriving in both wastewater and agricultural soil25. The relative abundance increases for these phyla may suggest that bacteria in wastewater could become part of the soil microbes through the use of irrigation. A following result may be the decrease of the other phyla induced by competition . In addition, these effects of introduced microbes may be related not only with the interference that exogenous populations may have on the soil microbial community, but also with the capacity of the exogenous organisms to survive in soil and constitute a health risk to soil ecological system and quality25.Differently, the richness and diversity of bacterial communities were all significantly higher in AWS. PCA also showed that bacterial communities were clustered in FWS and random in AWS. This pattern is consistent with the result of Li 41. Under this circumstance, the observed reduction of functional diversity in this study may attribute to the decreases in the taxonomic diversity of specialist species in Proteobacteria, Bacteroidetes and Planctomycetes while the increases in the Actinobacteria, Chloroflexi, Acidobacteria and Gemmatimonadetes could not increase the taxonomic diversity of specialist species. In addition, consequences of such shifts may also change some specialized soil functions. As Smalla et al.42 discovered, by comparing the temperature gradient gel electrophoresis (TGGE) profiles of the original inoculum with those in the BIOLOG wells following incubation, that fast-growing bacteria which adapted to high substrate concentrations were numerically dominant in the BIOLOG wells. Therefore, the comparatively high diversities of specialist species may more accurately reflect the functional community. In this study, however, as the patterns of substrate utilization only indicated functional aspects of the culturable fraction of the community inoculated to the wells, the shifts in bacterial community composition may be less well represented the functional changes, thus, future work would be needed for more accurate assessment.Based on the above results, we found that although the soils irrigated with aquaculture wastewater exhibited higher bacterial community richness and diversity Table , they sh4) concentration in soils while the variation of bacterial community structures was mainly correlated with P, EC and ions  concentration. Generally, aquaculture wastewater tend to be very saline (15 dS m−1 < EC < 45 dS m−1) and often have higher nutrient levels, such as phosphorus and nitrogen38. Although muds desorption (used in our sampling region) as a physical chemical water treatment technology was observed to be effective in removing phosphorus43, soluble salts left in wastewater would still inevitably promote soil salinisation by irrigation, which in fact was the most commonly reported negative effects of wastewater irrigation45. The positive relationship between phosphorus and microbial functional diversity had been reported by many studies47. As the soil total phosphorus decrease was indeed detected in our study (from 10.72 in FWS to 7.87 mg kg−1 in AWS), the reduction of soil microbial functional diversity and their significant correlation could be understood. Majority previous studies had concerned the negative impacts on soil microbial activities as soil salinity increased50. Jin et al.51 found AWCD values decreased significantly with increasing soil salinity. Setia et al.49 reported that saline soil  reduced soil respiration even by more than 50%. However, the effects of soil salinity or salt ions on microbial community structure are still fragmented and incomplete, especially based on metagenomic methods52. Our result demonstrated that bacterial phyla Bacteroidetes had completely different response to salt ions compared with OD1, although they were all sensitive to salt ions concentration changes. As the correlation analysis used in this study were just based on two treatments including 6 samples, more work should be done in future to clearly distinguish the response of different microbes to salt ions. Increased soil Cl concentration induced by aquaculture wastewater irrigation were detected playing a primarily role in the reduction of microbial functional diversity and the variation of bacterial communities in our study. It is not surprising as salinity toxicity is often attributed to the Cl that is massively present in saline soils54. Zahran55 found that the microbial growth was significantly inhibited by the toxicity of Cl. Gryndler et al.56 also detected Cl concentration would mainly affect indigenous microorganism. Many biogeochemical processes would also be influenced by soil Cl concentration, such as nitrification, which was reported inhibited by chloride salts57. These results demonstrated that more attention should be paid to soil Cl concentration control in order to alleviate negative effects of aquaculture wastewater irrigation on soil ecosystem.According to the relationship analysis Table , it appe2 areas and bred about 8,000 fish in 2014. Around the station, grapes were planted in more than 10 ha farmlands. A lake , the water source of which was Qilian glacial snowmelt, supplied water for fish breeding and the most of grape fields irrigation. The wastewater from all fish ponds were collected, muds-adsorption treated and transported into designed grape fields near the station. The chemical composition of lake fresh water and treated wastewater were shown in Table In order to undertake the present study, a cold-water fish breeding station, which is located in Yangguan Town, northwest China  was chosen as the sampling region. As sit at the boundary of Kumtag Desert, the mean annual precipitation of this region is only 39.9 mm and the annual temperature is 9.3 °C. The station covered approximately 30,000 mth, 2014. One kind was irrigated with water from Moon Lake (fresh water irrigated soil) and another with aquaculture wastewater. In each kind of field, 3 sites were chosen as duplicates of sampling. Totally 13 non-rhizosphere soil cores (0–20 cm depth) were collected from each site according to an S-shaped curve, and then completely mixed into one composite soil sample. After sieving out plant roots and stones, 1 kg of soil sample was obtained and put into a sterile bag. Totally 6 soil samples were obtained and packed in ice blocks and then transported to laboratory within 24 h, where each sample was divided into three parts. One part was refrigerated at 4 °C for microbial functional diversity analysis, a second part was frozen at −80 °C for microbial genetic analysis and others were air dried for chemical analysis.Soil samples were collected from two kinds of grape fields on July 1658. Total nitrogen (N) was measured using the Kjeldahl method59. Total phosphorus (P) was determined by NaHCO3  extraction60. The pH and EC were determined in a soil suspension with deionized water (1:5 w/v). Detailed salinity  was measured by the ion exchange chromatography . The results were shown in Table Soil organic carbon (C) was analyzed by colorimetry after oxidation with a mixture of potassium dichromate and sulphuric acidTM. The plates have 96 wells and each plate consisting of three replicates (comprising 31 sole carbon sources and one water blank). In this study, 5 g of each soil sample was suspended in 45 ml of sterile saline solution (0.85% NaCl) and shaken 30 min on an orbital shaker. Then 1 ml of soil suspension was transferred into a microcentrifuge tube and centrifuged at 10,000 rpm for 20 min. The supernatant was removed. The pellets were washed twice to remove water soluble carbon using the sterile saline solution and resuspended in 20 ml of the same solution. A 150 μl sample of the suspension was inoculated into each well. The plates were incubated at 25 °C. Color development in each well was recorded as optical density at 595 nm and 750 nm at 24 h intervals for 168 h.Soil microbial metabolic activity was measured using Biolog Ecoplates61. Microbial activity in each microplate, expressed as AWCD value was determined according to Garland and Mills62. The 168 h optical density value  for each sample in triplicate, divided by their AWCD to normalize the values, was used to calculate the utilization of carbon sources63. Indices of the functional diversity (Shannon and McIntosh) were calculated according to Gomez et al.64.The well absorbance values were adjusted by subtracting the absorbance of the control well. The final values in each well were the 590 nm values minus the 750 nm values. Negative readings were set to zeroFor each soil sample, the DNA was extracted using a Power Soil DNA Isolation Kit  according to the manufacturer’s instructions. Briefly, 0.25 g of soil was loaded into bead tubes containing solution C1, incubated for 10 min at 60 °C and then vortexed for 10 min at maximum speed with the MoBio vortex adapter. The manufacturer’s protocol was followed from this point onward. Following elution, DNA samples concentrated by ethanol precipitation and resuspended in 10 mM Tris. The extracts were assessed for quality and quantity using a spectrophotometer and checked for integrity by 0.8% agarose gel electrophoresis. The V4 regions of the bacterial 16 S rRNA genes were amplified using primers 520 F (5-AYT GGG YDT AAA GNG-3) and 802R (5-TAC NVG GGT ATC TAA TCC-3). After quantified using Quant-iT PicoGreen dsDNA Assay Kit, the PCR products were mixed and sequenced using the 454 GS-FLX Titanium system at Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China.65 with default quality settings. They were filtered if they had an average quality score lower than 25, contained fewer than 150 bases and had ambiguous bases. Subsequently, chimeras were checked and eliminated by the MOTHUR program with the UCHIME algorithm67. Then, the high-quality sequences were clustered into OTUs at 97% identity using the UCLUST algorithm. The consensus sequence of sequences in each OUT was used as a representative sequence. The taxonomic identity of each representative sequence was assigned to bacteria by using the RDP classifier68 trained Greengenes reference database.The raw sequence reads were denoised using a standard QIIME -based pipeline4 sequences from each soil sample. Based on 97% sequence similarity, 4 133 bacterial OTUs were identified, with a median of 3 399 OTUs in FWS and 3 612 OTUs in AWS (Table 66.We obtained 88 052 high-quality bacterial 16S Vt- test for two independent samples was performed to detect the significant difference using the SPSS 18.0. Heatmap was generated by R packages to exhibit the 31 carbon substrates utilization. PCA was used to identify the microbial characteristics of each sample. Multivariate tests were carried out using PERMANOVA on Euclidean distances with the Adonis command in the vegan package of R. RDA was conducted for analyzing the correlations of microbial characteristics and soil chemical variables. Before the RDA, we conducted selection of the soil variables using the stepwise regression method and the Monte Carlo Permutation test. PCA and RDA analyses were conducted using Canoco 5.0.A paired