Gait and waist motions always contain massive personnel information and it is feasible to extract these data via wearable electronics for identification and healthcare based on the Internet of Things (IoT). There also remains a demand to develop a cost‐effective human‐machine interface to enhance the immersion during the long‐term rehabilitation. Meanwhile, triboelectric nanogenerator (TENG) revealing its merits in both wearable electronics and IoT tends to be a possible solution. Herein, the authors present wearable TENG‐based devices for gait analysis and waist motion capture to enhance the intelligence and performance of the lower‐limb and waist rehabilitation. Four triboelectric sensors are equidistantly sewed onto a fabric belt to recognize the waist motion, enabling the real‐time robotic manipulation and virtual game for immersion‐enhanced waist training. The insole equipped with two TENG sensors is designed for walking status detection and a 98.4% identification accuracy for five different humans aiming at rehabilitation plan selection is achieved by leveraging machine learning technology to further analyze the signals. Through a lower‐limb rehabilitation robot, the authors demonstrate that the sensory system performs well in user recognition, motion monitoring, as well as robot and gaming‐aided training, showing its potential in IoT‐based smart healthcare applications. A waterproof and textile‐based triboelectric tactile sensor is designed for healthcare applications including motion intention detection for robotic control, human–machine interaction‐based waist training, and gait analysis. Combined with machine learning and Internet of Things technology, all these functions are integrated and demonstrated with a lower‐limber rehabilitation robot to amplify its functionality and improve training outcomes. Among them, various wearable sensors are attached to the skin or worn on the body directly for sensory information collection with promising results distinguishing distinct body behaviors. For instance, the smart socks with deep learning‐enabled signal processing and the exoskeleton‐shaped multiple degrees of freedom sensory system were developed for gait analysis and posture perception, respectively. Such wearable sensory system enables the digitalization of human activities to create data‐based insight into health status and activity management, bridging the gaps between the human and machines. In the meantime, with the remarkable progress of fifth‐generation wireless networks, the delay of the information transmission can be ignored and all the sensory information could be uploaded to the cloud for remote analysis and visualization via Internet of Things (IoT) and big data. Generally, wearable sensors together with seamless data exchange for human motion detection will contribute to smart healthcare applications.Wearable or flexible electronics emerges as a rapidly developing research field in recent decades because of its intrinsical flexibility and lightness.1, 2 For ambulation‐impaired patients, the sensitive and friendly HMI to achieve the dexterous and accurate manipulation of the lower‐limb rehabilitation robot is of vital importance. Note that the waist always cooperates with the near lower limbs to maintain the walking balance, and provides valuable information of the motion intention. Thus, the HMI devices designed for waist motion capture can provide the sensory information to control the robot to follow and assist the patient's walking. In terms of physical sensing techniques, the vision recognition and initial measurement unit (IMU)‐based HMIs are still the widely adopted solutions to distinguish human motions for robotic manipulation. However, the vision recognition by cameras may cause privacy issues, while bulky IMU networks with a lack of flexibility lead to the discomfort. Furthermore, complicated calculations and high computing power are normally necessary, resulting in sizable external circuits and components. Alternatively, a flexible sensor transferring motion states into electrical signals in a more straightforward way reveals its merits of minimizing the sensory structure. Besides, with the rapid development of virtual reality (VR) and augmented reality (AR), some recent works focus on gaming‐based HMIs via flexible wearable sensors to facilitate the user to absorb in the virtual space. Such intelligence and functionality‐enhanced HMIs pave a new way to motivate the training with immersive gaming control to relieve the weariness and gain enjoyment during long‐term rehabilitation.Disorders of gait and posture are debilitating and common, and adequate monitoring of those disorders is imperative as it can provide useful clues to the underlying pathology in patients and the recovery progress of various medical conditions. Meanwhile, lower‐limb rehabilitation robotic has been proposed as an effective tool to restore the economy of bipedal gait.28 For Till now, several basic approaches are adopted for diversified HMIs and robotic perception, including conductive nanocomposites, capacitive variation, piezoelectric or triboelectric effect, etc. For instance, a cutaneous mechanoreceptor using capacitive variation was developed for soft machines, physiological sensing, and amputee prostheses, showing its outstanding sensitivity and wide pressure range. Wang et al. proposed a multifunctional tactile sensor via a hierarchically patterned structure for multiple stimulus detections. In general, they exhibit advances in accuracy and stability, but some of the aforementioned methods are still restrained by several drawbacks, e.g., resistive sensor usually suffers the temperature drift and the parasitic capacitance affects the sensing result for the capacitive sensor. Moreover, the resistive or capacitive sensors must consume considerable electrical power to maintain their operations and they tend to require complicated chemical or physical treatment for fabrication, leading to energy and cost issues for the massive distributed sensory network. In addition to sensing performance, the flexible wearable sensor is also in high demand of properties including self‐sustainability, ease of fabrication, and low cost for customizable applications. On that account, the triboelectric nanogenerator (TENG) featured with self‐generated signals and lower power consumption is attracting increasing attention widely, especially for the scenarios of large‐scale IoT applications. More importantly, due to the unique operation mechanism, triboelectric sensors have versatile options in terms of fabrication techniques and materials.In terms of flexible wearable sensors, the remarkable process has been made with different structures and materials in the past few years.41 Til There are a few efforts in developing textile‐based TENG sensors for healthcare applications, which exert superb capabilities of structural retention and fatigue resistance, during long‐term wearing and washing. For example, He et al. reported a narrow‐gap TENG based on textiles for harvesting low‐frequency and irregular waste energy from body motions to power a Bluetooth module, rendering a self‐sustainable temperature and humidity monitoring system. However, such kind of structure integrated with garments is not suitable for robot‐aided lower‐limb rehabilitation in need of gait detection and waist motion capture since the sweat will cause the unexpected performance change during the long‐time on‐body sensing. In consideration of that, the special design for stabilizing the working condition, especially for humidity, becomes extremely important. Note that the thermoplastic polyurethanes (TPU)‐coated fabric usually applied in medical fields like sphygmomanometer features water resistance without the sacrifice of flexibility and thus, this material may provide a promising solution to maintain TENG's performance through facile heat sealing of the TPU layer. In addition, artificial IoT (AIoT) techniques can be further utilized to amplify the intelligence capabilities of wearable devices via efficient communication with the cloud, and it can extract the higher‐level information from the raw data to simplify the signal processing and compensate the errors.Textile, as a fundamental part of normal clothes, is well suitable for wearable devices benefiting from its unique properties of light weight, soft nature, and wearable comfortability.67 TheFigure Herein, we report a textile‐based triboelectric sensory system for gait analysis and waist motion capture to enhance the intelligence and performance of the robot‐aided lower‐limb and waist rehabilitation Figure 1a,b. Sin2Figure Though the TENG technology offers a facile and low‐cost fabrication process for flexible wearable sensors, the sensitivity and sensing range are required to be considered since there always suffer distinct pressures during monitoring gait and waist motion. Also, building up a water‐resistant structure is of vital importance for the scenarios of long‐time and high‐intensity training as the sweat will lead to voltage variation. Here, we adopt the encapsulated narrow‐gap structure with pyramid patterns distributed on the negative triboelectric layer to construct the device Figure 2a. The TTo characterize the electrical output performances of the TENG‐based sensor, a multi‐material test machine is employed to adjust the compressing pressure and speed Figure .Considering the TENG‐based sensor also has grand potential in energy harvesting, its output performance is measured with various external load resistances from 0.1 to 1000 MΩ Figure . Accordi3 The device for waist rehabilitation becomes significantly important because the health of the waist is one of the major factors to maintain the life quality. However, weariness and inattention hinder the long‐term waist training, and there remains a demand to improve the enjoyment and motivation for a better rehabilitation outcome. Meanwhile, benefiting from the drastic advancement of AR/VR technologies, the promotion of diversified HMIs paves a new way for immersive healthcare programs via the virtual game, digital twin, robotic manipulation, etc. Hence, in order to construct the HMI for waist rehabilitation apparatus, four textile‐based TENG sensors are sewed onto the waist safety belt equidistantly to form W‐TENGs. As depicted in Figure In modern society, with the rapid development of technology, intelligent machines tend to replace heavy physical labor and people start to break away from the traditional work style. Alternatively, they remain seated and immobile staring at computer monitors for a long time, leading to the high risks of waist injuries.75 TheBased on such sensory method, the intelligent safety belt‐based HMI together with rigid bracket to support the user is implemented to realize waist rehabilitation enhanced by vehicle control, as shown in Figure 4 For instance, in items of the robot‐aided lower‐limb rehabilitation, as the patient takes walking training, the necessary information can be digitalized to explain the step number, walking speed, etc., enabling to regulate the amount of training and analyze the rehabilitation status. Currently, two basic concepts are adopted to accomplish this task, i.e., sensitized insole or sock. In this work, a couple of textile‐based TENGs marked I‐TENG‐1 and I‐TENG‐2 are fabricated and arranged onto the front and back sides of the insole, respectively, to improve the intelligence for gait analysis, as shown in Figure Gait always contains valuable sensory information and it makes sense to monitor the gait via distinct sensors to collect the diverse physical parameters.79 ForVs = L/T, where T is the time interval between two positive voltages for heel contact, and L is the stride of one walking cycle. As shown in Figure To further verify the reliability of smart insole‐based gait analysis, some extra experiments have been conducted by the same volunteer. As shown in Figure To explore the signals except for four‐state walking on the flat ground, the TENG signals for jumping, going upstairs, and going downstairs are measured. As shown in Figure 4f, unlike normal walking with sole contact and separation, the jumping suffers more complicated signals, which can be divided into state (i) and state (ii). The reason for this phenomenon is that the jumping undergoes energy storage and landing steps, and the foot leaves and contacts the ground at the same time. Meanwhile, signals for going upstairs or downstairs possess their specific characteristics as well, e.g., the heel and toe contact at the same time for going upstairs and the toe tends to stand first to ensure safety for going downstairs. Depending on the sensitivity of the smart insole, we apply it for Parkinson's disease (PD) and fall detection to alarm for the emergency situation during the rehabilitation process. PD is a progressive neurodegenerative movement disorder that often manifests as gait disturbances and even falling. However, PD is difficult to be evaluated during clinical examination because it is usually triggered by uncertain or specific conditions, resulting in the significance of monitoring patients with PD. In addition, for these elderly patients or the disabled, the capability of fall detection is of vital importance to guarantee human life. To reflect the possibility to utilize the smart insole in these applications, the mimetic motions of falling and PD are performed, and the corresponding detected signals can be found in Figure 5 Gait implicates multi‐layer characteristics  and these privacies always couple together to form facile and natural encryption via a set of triboelectric signals using the proposed smart insole. Unlike traditional analysis strategies to obtain the shadow features such as frequency and peak number, recent emerging machine learning as a technique for extracting nuances has been used for data acquisition and pattern analysis of triboelectric signals, providing the possibility of integrating artificial intelligence (AI) technology with wearable electronic technology to construct a complete intelligent system. Moreover, this approach can comprehend subtle information for complex features in a higher dimension which cannot be resolved by naked eyes after training the model. Among the machine learning methods, artificial neural network (ANN) is a quite effective approach to solve complex classification problems with good robustness and scalability, and it has been proposed to be applied in analyzing tactile signals for high‐accuracy object recognition. Thus, the smart insole combined with ANN‐based predicting model provides an alternative solution for patient recognition to fulfill the scenarios involving information identification, avoiding the risks of privacy disclosure.In this era of information expansion, there arises the privacy concern of the face, fingerprint, and acoustics recognitions because of their massive personnel information.83 GaiFigure Meanwhile, we insert dropout and batch normalization layers into the first several FCLs to enhance the robustness and accelerate the training speed, while the last FCL outputs the predicted identifiers of the five participants  to extract features from the triboelectric signal to realize the patient recognition for rehabilitation plan selection Figure5a,b. ToIn terms of the healthcare system, the expensive medical devices such as the rehabilitation robot are frequently shared by multiple patients and there usually occurs privacy issues as the training data including personal information are always left in the machine, bringing in the motivation for patient recognition and constructing accessible private accounts in a feasible and user‐friendly manner. Based on the machine learning model, we extend the identity recognition into the rehabilitation system, and thus, the customized training plan can match with different patients via the IoT‐based distant setting. Note that the sole 100‐sample training data are proved to realize a high recognition accuracy, indicating the significance and possibility for the disabled patient to follow this identification. We demonstrate the patient recognition based on the established model for the same five participants and the result indicates that the system accomplishes the function to access their personal accounts for training plan selection  and it is demonstrated to achieve multiple functions including user recognition, rehabilitation monitoring, robotic manipulation as well as gaming‐enhanced training. In general, the proposed triboelectric sensory system provides a low‐cost, energy‐saving, and universal solution for the realization of the AIoT‐based smart healthcare system in near future.8The pyramid‐patterned triboelectric layer was made of silicone rubber and it was fabricated through the mold designed by Solidworks 2019 and printed by a 3D printer  with the filament . The required amount of part A and part B of the Ecoflex 00–50 (Smooth‐on) was dispensed into a container (1A:1B by weight) and stirred for 1 min before the material was vacuumed for 3 min to reduce the bubbles. Then, the mixed solution was poured into the 3D‐printed mold followed by a 30 min baking at 70 °C for curing. The PES fabric, a kind of high‐fiber stitched textile, and TPU film were cut into 50 mm × 50 mm, while the conductive textile  sized 35 mm × 35 mm was prepared to serve as both the electrodes and electrification materials. They were composited together to form 0.4 mm thickness TPU‐coated fabric via electronic iron with about 160 °C, where the hot‐melt TPU film in the middle acted as the adhesive. After gluing the silicone layer onto the Ni‐fabric face of the composite textile with the liquid silicone, a thermal sealer  was utilized to bond the TPU film's marginal area to seal the TENG structure.The open‐circuit voltage, short‐circuit current, and transferred charges were measured by an electrometer  and recorded by an oscilloscope . The measurement for TENG signal optimization was realized by the same oscilloscope with a 100 MΩ impedance probe. The required sensing range for both gait and waist motion detection was estimated by arranging the commercially available sensor  onto the safety belt and insole. In order to validate the sensor output for different application scenarios, a multi‐material test system  was accepted to regulate the compressing force and speed. Besides, the TENG‐based sensor was heated by the heating plate  to verify its performance under distinct thermal conditions.Two and four as‐fabricated triboelectric sensors were sewed onto the insole and safety belt, respectively. The real‐time signals for all the applications were collected by the MCU  with the signal processing circuit. The commercial Bluetooth module (HC‐05) was utilized to transmit the data in the vehicle control application. The ANN‐based machine learning system was developed in Python with Keras and Tensorflow frameworks. The robot‐aided rehabilitation was conducted by integrating the triboelectric sensory system into the low‐limb rehabilitation robot (iReGo) capable of supporting the user to walk.Prior to participation in the experiments, informed consent was obtained from the volunteer in all experiments.The authors declare no conflict of interest.Supporting InformationClick here for additional data file.Supplemental Video 1Click here for additional data file.Supplemental Video 2Click here for additional data file.Supplemental Video 3Click here for additional data file.Supplemental Video 4Click here for additional data file.Supplemental Video 5Click here for additional data file.Supplemental Video 6Click here for additional data file.Supplemental Video 7Click here for additional data file.


Adipose‐derived mesenchymal stromal cells (AdMSC) release numerous soluble factors capable of stimulating angiogenesis. Improved methods for delivering these cells to maximize their potency are now sought that ideally they retain viable cells in the target tissue while promoting the secretion of angiogenic factors. Substrate surface topography is a parameter that can be used to manipulate the behavior of AdMSC but challenges exist with translating this parameter into materials compatible with minimally invasive delivery into tissues for in situ delivery of the angiogenic secretome. The current study investigates three compositions of hierarchically structured, porous biodegradable microcarriers for the culture of AdMSC and the influence of their surface topographies on the angiogenic secretome. All three compositions perform well as cell microcarriers in xeno‐free conditions. The attached AdMSC retain their capacity for subsequent trilineage differentiation. The secretome of AdMSC attached to the microcarriers consists of multiple proangiogenic factors, including significantly elevated levels of vascular endothelial growth factor, which stimulates angiogenesis in vitro. The unique properties of hierarchically structured, porous biodegradable microcarriers investigated in this study offer a radically transformative approach for achieving targeted in vivo delivery of AdMSC and enhancing the potency of their proangiogenic activity to induce neovascularization in ischemic tissue. This study investigates highly textured microspheres designed for use as implantable cell microcarriers that are shown to stimulate the secretion of proangiogenic factors from attached mesenchymal stromal cells. These findings suggest that the technology can provide a feasible and transformative new approach for treating cardiovascular disease through the targeted in situ delivery of factors that will promote new blood vessel growth. In relation to cardiovascular disease, the MSC secretome has been reported to influence angiogenesis in vitro and in vivo, facilitating revascularization, immune modulation, wound healing and tissue repair. Due to the potent trophic and paracrine immunomodulatory activity of MSC, there is an increasing interest in exploiting the MSC secretome for new therapeutic strategies  secrete a plurality of molecules, collectively called the MSC secretome, which include a heterogeneous pool of soluble proteins, free nucleic acids, lipids and extracellular vesicles that have effects on different cell populations (reviewed in ref. ). The MS in ref. , 9). or lipopolysaccharide can be sensed by MSC and subsequently alter their paracrine activities. Furthermore, physiological manipulation, such as exposure to hypoxic conditions, has been shown to increase secretion of certain growth factors, such as vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF‐2), hepatocyte growth factor (HGF) and anti‐inflammatory molecules  of MSC results in a significant enhancement of their proangiogenic paracrine activity compared with unstimulated MSC. Increased understanding of the physicochemical cues that influence the MSC secretome is a prerequisite to harnessing the paracrine activity of these cells for therapeutic applications in cardiovascular disease, cancer, neurodegenerative diseases and regenerative medicine.The paracrine activity of MSC is affected by the local cellular microenvironment, which comprises a plethora of biochemical, mechanical and physical cues (reviewed in ref. ). Solubl in ref. ). The mo through to the development of elaborate cell culture platforms with imprinted physical cues used to study the mechanisms underpinning environmental sensing by MSC and control of their behavior. More recently, investigators have started to study the MSC secretome in response to such cues. However, to date, there has been a paucity of new technology compatible with easily delivering these cues in vivo and that is compatible with existing, scalable platforms used in bioprocessing to ease future translation.Biomedical engineering has made possible the development of novel cell culture substrates that provide instructive cues at a cellular and subcellular level and has highlighted potential new opportunities for harnessing the therapeutic effect of MSC. Examples include cues on 2D substrates, such as topographical, geometrical and mechanical features that affect MSC growth and lineage specification, Cell attachment to microcarriers can be enhanced by inclusion of instructive cues, such as physical features  and biochemical properties , as reviewed elsewhere). However, existing commercial microcarriers are not suitable for clinical translation. Therefore, significant opportunities exist for the creation of new, smart biomaterial‐based microcarrier substrates that combine cell instructive cues in a format that can be integrated with the extensive technology of that already exists for culturing microcarriers and are biocompatible for in vivo implantation. Such technology would deliver a transformative approach for targeted 3D substrate‐induced manipulation of the MSC secretome in situ.Polymeric microcarriers are widely used as cell substrates during the expansion of MSC in scale‐up bioreactor systems. All three TIPS microcarrier types performed well as cell microcarriers, while cell attachment of AdMSC on solid microparticles made of the same polymer was very limited. Data from the study demonstrate that attachment and culture of human AdMSC on the surface of TIPS microcarriers in xeno‐free conditions results in an enhanced proangiogenic secretome. This finding may provide a new approach for enhancing the paracrine activity of MSC for use in revascularizing ischemic tissue.The current study reports for the first time on biodegradable microcarriers with unique hierarchical porosity designed to manipulate MSC toward a proangiogenic secretome. Three compositions of microcarriers with distinct surface topographies were fabricated using thermally induced phase separation (TIPS), a process that enables infinite adjustment to the hierarchical surface topography.22.1 In the current study, three different compositions of Poly‐d,l‐lactide‐co‐glycolide (PLGA) were used to create three types of TIPS microcarriers . All three polymers produced microcarriers that exhibited hierarchical pore structure characteristic of the TIPS process, but each exhibited different features of surface topography and porosity .The porous biodegradable TIPS microcarriers were fabricated using thermally induced phase separation. The manufacturing process has previously been shown to be simple, robust, and highly tunable, enabling control of size, surface topography, and degradation rate.y Figure. TIPS mi1 Figure . In compThe mean diameter of all three types of TIPS microcarriers was ≈300 µm Figure . TIPS miSolid PLGA microcarriers were included in the study as nontextured controls. All three compositions of polymer were compatible with the emulsion solvent evaporation technique for producing solid microcarriers; however, polymers 2 and 3 were most suited for achieving solid microcarriers similar to the size range of TIPS microcarriers .For the angiogenic secretome screening experiment, solid polystyrene (PS) microcarriers  have been used as additional control. The size range of these microcarriers was 250–355 µm (mean diameter 306.2 ±2 9.6 µm).2.26 AdMSC were cocultured with ≈17 500 TIPS microcarriers under semidynamic incubation conditions. Within the first 6 h, cells attached and began spreading on the surface of all compositions of TIPS microcarriers . There were no statistically significant differences among the three types of TIPS microcarriers.Measurement of lactate dehydrogenase (LDH) activity in the supernatants collected at different time points indicate the level of cell viability remained high throughout the study period and was comparable to the level measured for cells cultured on tissue culture plastic Figure . CompariAfter 11 days in culture, MSC that had migrated from the microcarriers onto tissue culture plastic retained their lineage plasticity and could differentiate toward adipogenic, osteogenic and chondrogenic lineage .2.4FigureThe concentration of VEGF was higher in supernatants collected from each composition of cellularized TIPS microcarrier compared with the supernatants collected from cells cultured on tissue culture plastic at all timepoints Figure. HGF, platelet derived growth factor (PDGF‐AA), placental growth factor‐1 (PIGF), IL‐1β and VEGF were increased in response to AdMSC culturing on the surface of TIPS microcarriers compared with AdMSC cultured on control microcarriers composed of solid polymer 3 or PS ,S Figure .FigureTo evaluate whether the secretome from cellularized TIPS microcarriers was proangiogenic, the collected supernatants were further tested in vitro using an angiogenesis assay that enabled quantification of capillary‐like tubule formation. At day 14, tubule formation was visible in wells incubated with supernatants collected from all compositions of the cellularized TIPS microcarriers Figure. The supTo investigate the potency of supernatants collected from a single time point, a parallel angiogenesis assay was conducted using supernatants collected from TIPS microcarriers composed of polymers 1 and 3 at day 3. The supernatants collected from both cellularized microcarriers at the single time point resulted in the formation of a greater number of tubules and junctions Figure  compared33.1The paracrine action mediated MSC represents an attractive tool for a range of cell‐based therapies. While many clinical studies have endeavoured to utilize this effect, existing modes of application are limited to delivering the inherently anchorage dependent cell type in a suspended formulation, detached from a substratum. A radically different approach for harnessing the paracrine secretion of MSC would consist of delivering the cells in a more natural state, i.e., attached to an implantable substrate, which would also provide opportunities to refine the surface to include cues that enhance specific components of the secretome. It is therefore feasible that PLGA TIPS microcarriers could be translated into clinical use for targeted delivery of AdMSC and to provide an effective stimulus for the secretion of proangiogenic growth factors. This would offer a transformative approach to harness the effect of the AdMSC secretome in a therapeutic setting.To explore this concept, the current study investigated whether highly porous, biodegradable PLGA TIPS microcarriers could be used for culture of AdMSC and manipulation of their secretome toward proangiogenic activity. A key feature of the microcarriers investigated in the current study is their potential for use as an implantable cell substrate. PLGA has an excellent clinical safety record and has been successfully used in a range of medical devices and drug delivery systems. TIPS microspheres have developed as a scaffold device for repair of perianal fistulas and are currently under clinical investigation.Compared with other techniques for the fabrication of porous microspheres, the TIPS process is simple, robust and does not require the addition of porogen material. The size and morphology of TIPS microcarriers produced for the investigation were readily controlled by parameters related to the polymer solution  and to the process . The method described in the current study enabled the fabrication of biodegradable microcarriers from three different compositions of PLGA that exhibited visibly different surface topographies. TIPS microcarriers of polymer 1 were highly porous compared with the microcarriers of polymers 2 and polymer 3. Microcarriers composed of polymer 1 underwent a greater reduction in size following the wetting process used to facilitate cell attachment compared with microcarriers composed of polymers 2 and 3. However, all three types of TIPS microcarriers were found to support equivalent cell adhesion and growth of AdMSC, with no significant difference observed between any of the microcarrier compositions. Similar phenomena account for the need to apply surface coatings to commercially available nontextured microcarriers to enhance cell attachment. The cell attachment and spreading behavior observed with TIPS microcarriers aligns with the notion that textured surfaces are conducive to supporting cell attachment and spreading.After 1 day of culture, the surface of the TIPS microcarriers were fully covered by a monolayer of AdMSC. On the contrary, AdMSC failed to adhere uniformly to the surface of the solid PLGA microcarriers that exhibited a smooth surface. The importance of surface topography on cell attachment is well recognized for other cell culture platforms. For example, the impact of surface topography on cell adhesion to polymeric microspheres has been described elsewhere, with limited cell adhesion occurring with solid microspheres compared with the microspheres exhibiting a textured surface. and stiffness also regulate lineage specification of MSC. On the contrary, the impact of the extracellular environment on the MSC secretome is an emerging field. Among the various cues that affect the paracrine activity of MSC, biochemical cues are better studied and understood. The effect of physical cues such as the topography of the cell substrate on the paracrine activity remains limited. Biomaterials that simulate physical cues experienced by MSC in their native environment might offer great potential of manipulating their secretome over conventional planar 2D cell culture substrates such as tissue culture plastic. Evidence in support of this concept includes preformed electrospun scaffolds composed of fibers in random, aligned and mesh‐like patterns that resulted in AdMSC producing significantly higher levels of anti‐inflammatory and proangiogenic factors compared with cells cultured on tissue culture microplates. Similarly, a comparison of flat substrates and 3D PS substrates resulted in differences in the crosstalk between MSC and endothelial cells or MSCs and human osteoblasts. Such studies suggest a biomaterial‐based approach is feasible for manipulating the paracrine activity of the MSC and, thus, increase opportunities for therapeutic applications. However, to be practicable for the broad range of uses where MSC are being investigated for their paracrine activity, rather than tissue replacement strategies, new approaches compatible with minimally invasive delivery are required. Implantable biodegradable microcarriers provide an ideal solution for this, since they provide a temporary cell substrate for delivery of cells in their anchored, natural state. Moreover, unlike other preformed scaffolds, the physical format of implantable microcarriers provide a formulation that can be delivered minimally invasively and conforms to the shape of the implant site.The cell substratum plays a key role in controlling MSC differentiation. Besides biochemical soluble cues, there is increasing evidence that physical cues, including topographical featuresIn the current study, a trend toward greater proangiogenic secretome was observed when AdMSC were cultured on PLGA TIPS microcarriers that exhibited a textured surface compared with smooth surfaces exhibited by control PLGA microcarriers, PS microcarriers or PS tissue culture plates. Cells cultured on TIPS microcarriers secreted higher amounts of VEGF at all time points up to day 7 compared with cells cultured on tissue culture plastic. It is worth noting that even though the supernatants collected from cellularized TIPS microcarriers were diluted with endothelial growth medium prior to being applied to the in vitro angiogenesis assay, the quantity of VEGF present still exceeded the concentration of the VEGF positive control. The presence of proangiogenic factors in the supernatant collected from AdMSC cultured on TIPS microcarriers accounts for the increase in tubule formation and tubule junctions quantified in the angiogenesis assay. This was supported by the plurality of factors detected by the proteome profiler array. Several of the key proangiogenic factors found to be increased in the pooled supernatants are likely to contribute to the increased angiogenesis observed in vitro. In contrast, the level of multiple proteins in supernatants collected from solid microcarriers were found to be reduced despite being composed of the same polymer, indicating that the textured surface of the TIPS microcarriers rather than its chemical composition contributes to the effect observed. We speculate that the reason for the differences in secretome profile may relate to how AdMSC attached to the different type of PLGA microcarriers. AdMSC formed clusters at the surface of the solid microcarriers  and glycolate , which in vivo can be metabolized and excreted by the host. Lactic acid has been reported to be an important contributor to angiogenesis in wound healing and to stimulate endothelial cell migration in vitro.Overall, these findings support the notion that the proangiogenic secretome can be directly influenced by the physicochemical properties of the substrate and represents an important parameter to consider when designing implantable microcarriers. The current in vitro proof‐of‐concept study indicates that biodegradable porous microcarriers stimulate the secretion of proangiogenic factors from AdMSC capable of inducing angiogenesis. It is recognized that while the in vitro angiogenesis assay combined with data from the ELISA and proteome angiogenesis array provide a useful indication of this response, the results from this study will need to be validated using suitable in vivo models.Although higher values of VEGF were measured between days 1–7, the amount of VEGF measured in the supernatants collected at days 9 and 11 were similar between the TIPS polymer groups and the TC group. The reason for this is uncertain but might relate to changes to the physicochemical properties of the microcarriers as they start to degrade under hydrolysis. This is further supported by findings that when the in vitro angiogenesis assay was incubated with supernatant collected from cellularized TIPS microcarriers at a single timepoint, there was an increase in tubule formation compared with tubules formed when incubated with AdMSC secretome collected from different time points. This finding suggests that the temporal profile of the AdMSC secretome influences the angiogenic process. The mechanism underlying the change in composition of the AdMSC secretome might arise from either change to the texture of the TIPS microcarriers as the polymer degrades under hydrolysis during cell culture, which raises the prospect of further refinement and identification of specific surface topographies that induce optimum cell activation. Alternatively, temporal response may result of paracrine signaling pathways arising from the secretome influencing the behavior of the cells. The importance of a temporal control of angiogenesis is recently increasingly been emphasized.The stimulatory effect of the material did not appear to affect the phenotype of the AdMSC since cells attached to the surface of the PLGA TIPS microcarriers and subsequently migrated off retained their capacity for trilineage differentiation. This is an important finding since if the substrates led to lineage commitment it would limit future clinical use to implantation of cellularized microcarriers into tissues associated with that lineage. This observation, together with the tunable properties of TIPS microcarriers related to the choice of microcarrier polymer composition, porosity, rate of degradation and size, present further opportunities for tailoring implantable microcarrier substrates that can be designed to enhance the therapeutic effects of MSC mediated by the paracrine factors secreted by these cells, and warrants further investigation.Given the intended purpose of TIPS microcarriers is to provide an implantable substrate for adhered cells, the cellularized microcarriers were incubated under static conditions for extended periods in vitro to simulate the interplay between cellularized TIPS microcarriers when implanted as a bolus in vivo. Under these conditions AdMSC cultured on TIPS microcarriers readily formed neotissue constructs via cell–cell connections between adjacent microcarriers, demonstrating they are well suited to supporting cell growth. The formation of the tissue construct is likely to have provided a niche for the paracrine interaction of the plethora of factors released by the cells. The importance of cell–cell interactions on paracrine activity was highlighted recently by Qazi et al., where microporous scaffolds that promoted cell–cell interactions enhanced the activity of MSC compared with nano‐porous hydrogels that prevented cell–cell interactions.3.2This study provides new insight into the use of material‐based biosystems that can be used to harness and enhance the therapeutic potential of MSC. To verify the proangiogenic potency of the enhanced secretome observed in the current study it will be necessary to investigate the system using suitable in vivo models. This will provide a better understanding of how the secretome influences the complex multicellular processes associated with angiogenesis, as well as the influence of dynamic factors, such as microcarrier degradation and increased vascularization in the local environment, on MSC survival, retention and function. It will also be instructive to conduct longer‐term studies that enable the duration of effect to be observed beyond the time it takes for complete biodegradation of the microcarriers.Although the AdMSC used in our study are from a well‐characterized commercial source it will be interesting to investigate potential donor‐to‐donor variability and whether MSC derived from other tissues  behave in the same way. traumatic brain injury, and immunomodulation. It is therefore conceivable that an alternative application of the MSC‐TIPS microcarrier system might involve collection of cell‐free conditioned supernatants containing factors and extracellular vesicles from large‐scale bioreactors for cell‐free clinical application.It is generally accepted that the majority of MSC‐mediated therapeutic benefits relate to the secretion of bioactive molecules. Potential therapeutic benefit of cell free MSC‐conditioned supernatants has been suggested for a variety of conditions including cardiovascular disease,4The current study demonstrates for the first time that biodegradable hierarchically structured microcarriers suitable for in vivo delivery of AdMSC can be tailored to stimulate the secretion of a proangiogenic secretome. These findings offer a feasible approach for achieving targeted delivery of AdMSC and harnessing their proangiogenic effects to increase neovascularization, which could be used as a new therapy for cardiovascular disease.5 Briefly, PLGA was dissolved in dimethyl carbonate  using magnetic stirring overnight. Three different PLGA polymers that differ in the ratio of lactide:glycolide were used . Each polymer was dissolved at different weight:volume (w/v) ratios of DMC: 1% (w/v) PDLG8531 (polymer 1), 5% (w/v) PDLG5010 (polymer 2), and 10% (w/v) PDLG7507 (polymer 3).Hierarchically structured PLGA microcarriers were prepared using thermally induced phase separation (TIPS), as previously described.−1. The vibration frequency of the nozzle was kept at 2.70 kHz and the amplitude of frequency at 100%. The polymer droplets were delivered into a 1 L polypropylene beaker containing liquid nitrogen to achieve thermally induced phase separation of the polymer and solvent before being lyophilized in a freeze drier (Edwards MicroModulyo) for 24 h to achieve sublimation of the frozen solvent. The dried microcarriers were sieved to within a size range of 250–355 µm, which is a similar size range to commercially available microcarriers used for bioprocessing.Droplets of the polymer solution were generated using a Nisco Encapsulator Unit Var D (Nisco Engineering), fitted with a stainless steel, sapphire‐tipped nozzle (100 µm orifice). The polymer solution was fed into the encapsulator unit through silicone tubing via a syringe pump  at a constant rate of 2 mL min Accordingly, each polymer was dissolved at different weight:volume (w/v) ratios of DMC: 1% (w/v) PDLG8531 (polymer 1), 5% (w/v) PDLG5010 (polymer 2), and 10% (w/v) PDLG7507 (polymer 3). The PLGA solution was delivered dropwise into an aqueous solution of 0.5% (w/v) polyvinyl alcohol  under constant agitation with a rate of 500 rpm achieved by mechanical stirring. The solution was stirred overnight at room temperature followed by collection of the microcarriers after three washes in deionized water. The recovered microcarriers were dried at 37 °C and placed into a vacuum desiccator for storage until further use.An oil‐in‐water (O/W) emulsion solvent evaporation technique was used for the preparation of solid PLGA microspheres following published protocols.Samples of the PLGA microcarriers were mounted on aluminum stubs using adhesive carbon tabs and sputter coated with gold (Polaron E5000) and viewed using a Hitachi S3400N SEM at 5 keV. The images acquired were analyzed using an image processing algorithm  to determine the size and shape of the TIPS microcarriers, by evaluating diameter and shape descriptors, including aspect ratio and roundness from top view SEM images.The number of PLGA microcarriers per unit mass was quantified using a particle image analyzer . Parameters measured included the size and shape of particles using the technique of static image analysis. Due to the high electrostatic surface charge of the TIPS microcarriers, the microcarriers were suspended in cell culture medium prior to analysis. Six replicate measurements were collected per type of microcarrier and the mean number of microcarriers per unit mass was calculated.2 gas adsorption–desorption measurement at 77 K on a Quantachrome Autosorb‐iQC. Prior to measurement, the powder samples (>100 mg) were degassed at 40 °C overnight under dynamic vacuum to ensure minimal adsorbate. The specific surface area and pore size distribution were calculated from the isotherm using the BET and Barrett–Joyner–Halenda (BJH) method, respectively. The total pore volume was estimated from the adsorbing amount of N2 at a relative pressure, P/P0, of <0.995.The porosity of the TIPS microcarriers was investigated by NThe surface porosity of the TIPS microcarriers  was determined from top view SEM images. The wetting solution consisted of 10% (v/v) absolute ethanol in cell culture medium . Dry TIPS and solid PLGA microcarriers were transferred into 7 mL polycarbonate containers and 5 mL of the wetting solution was added. The microcarriers were incubated under rotation in the wetting solution at 37 °C. Successful wetting of the microcarriers was confirmed by their sedimentation in the container. The microcarriers were rinsed twice in fresh culture medium and immediately used for the in vitro experiments.TIPS PLGA microcarriers were hydrophobic and required prewetting before cell attachment, as previously described.StemPro human adipose‐derived mesenchymal stromal cells  (passages 2 and 3) were cultured in Alpha Minimal Essential Medium  supplemented with 5% (v/v) HPL (Cook Regentec).6 AdMSC (passages 4 and 5) were mixed with ≈17 500 prewetted PLGA TIPS microcarriers of the three different PLGA compositions  or solid microcarriers in low‐attachment six‐well cell culture plates . Cells were incubated in semidynamic conditions (30 s at 250 rpm h−1) on a microplate shaker  for 18 h. After 6 h, unattached cells were removed by rinsing in PBS and the cell–microcarrier combination was fixed in 4% formaldehyde in PBS followed by permeabilization in 0.5% (v/v) Triton X‐100 and staining with phalloidin  and Hoechst  for cytoskeletal and nuclear staining, respectively. Cells were imaged using an inverted fluorescence microscope (Leica DM16000B). Quantification of the number of cells attached to each microcarrier was performed using ImageJ. The number of nuclei per microcarrier was calculated using the “Cell Counter” plugin. Counts were acquired from three separate experiments with ≈60–70 microcarriers counted per experiment. Cell coverage was quantified by calculating the ratio of the cell surface area per microcarrier surface area.To evaluate cell adhesion to the microcarriers, 0.5 × 104 cells mL−1 cell was added to individual wells on the plate and incubated for the specified amount of time at 37 °C in 5% CO2.A hanging drop culture plate  was used to attach a precise quantity of cells to individual microcarriers. A 40 µL droplet of proliferation medium containing a single microcarrier and 5 × 106 AdMSC (passages 4 and 5) were seeded onto ≈17 500 microcarriers as described above for each of the three different PLGA TIPS microcarrier compositions. After 18 h, the cellularized TIPS microcarriers were transferred to 24‐well low bind plates and cultured for a further 11 days under static conditions. The confined conditions of the 24‐well plate resulted in the microcarriers forming a multi‐layered 3D structure. The quantity of cells attached the microcarriers at each time point was measured using an automated counter . As a control condition, AdMSC were seeded at 4.5 × 104 cells per well in 2 mL medium on standard PS tissue culture plastic (“TC”) and cultured for further 11 days under static conditions. The use of cell densities greater than this led to rapid confluency and monolayer detachment before the completion of the study. At predetermined timepoints  the supernatant from each well was collected for further analysis and replaced by fresh proliferation medium. The quantity of cells was measured in one sample at each time point by DNA quantification using the DNAeasy Blood and Tissue kit (QIAGEN). DNA concentration in the samples was measured at 260 nm wavelength using a Nanodrop 2000c spectrophotometer (Thermo Scientific).To evaluate the effect of extended periods of AdMSC culture on TIPS microcarriers, 0.5 × 10Viability of AdMSC attached to TIPS microcarriers was assessed using the live–dead cell staining kit . At day 11, cellularized TIPS microcarriers were incubated with the staining solution for 30 min. Cell staining was observed immediately under a laser scanning confocal microscope (Olympus TIRF). Viable cells were stained with the cell‐permeable live dye , fluorescing green. Dead cells were stained with both the cell‐permeable live dye and the cell nonpermeable ethidium homodimer‐1, fluorescing red.The level of cytotoxicity associated with incubating cells attached to the microcarriers was evaluated using the CytoTox 96 nonradioactive cytotoxicity assay , which measures LDH, a stable intracellular enzyme that is released into the culture supernatant following cell lysis. The assay was performed according to manufacturer’s instructions. Three biological replicates were used per timepoint and per condition.The quantity of human VEGF in the cell culture supernatants collected from cellularized TIPS microcarriers at different timepoints was measured by ELISA . Supernatant from cells cultured on tissue culture plates or TIPS microcarriers without cells were included as control samples. Three biological replicates were collected from each timepoint and condition.n = 3 wells per condition). The culture medium in each well was replaced every two days with either conditioned medium collected at 2 day intervals or with conditioned medium collected at day 3 only.The angiogenic activity of supernatants collected from cellularized TIPS microcarriers was determined using V2a Kit‐Vasculogenesis to Angiogenesis . The supernatants from cellularized microcarriers or control samples (cells cultured on tissue culture plastic) at different points were diluted 1:1 with V2a growth medium before addition to the culture (−1) and negative (Suramin 20 × 10−6m) control samples were included as recommended by the assay manufacturer (n = 2 wells per condition). At day 14, the cells were fixed in 70% ice‐cold ethanol and stained with mouse antihuman CD31 primary antibody and goat antimouse IgG‐alkaline phosphatase conjugated secondary antibody, according to manufacturer instructions. Images of the stained tubules were acquired 4× magnification using a stereomicroscope . Tubule formation was analyzed using Cellworks Image Analysis Software, AngioSys 2.0 (Cellworks).Positive  at days 1, 4, 7, and 10 and replenished with fresh culture medium. The collected supernatants were immediately frozen until further analysis. The relative amount of soluble angiogenesis‐related proteins in the pooled supernatants was measured using the Human Proteome Profiler Angiogenesis Array , following manufacturer’s instructions. The optical density from chemiluminescent signal of each analyte dot was calculated in ImageJ and the values plotted using GraphPad Prism .After 11 days incubation in low attachment 24‐well plates, cellularized TIPS microcarriers were transferred into 6‐well tissue cultures plates and incubated for 4–6 days to allow cells to migrate from the microcarriers onto the plates and reach 60–80% confluency. The migrated cells were detached using trypsin‐EDTA and seeded into 12‐well tissue culture plates at densities suitable for trilineage differentiation.4 cells cm−2 and cultured in MesenPRO RS Medium. After 3 days, the culture medium was replaced with StemPro Adipogenesis Differentiation medium (ThermoFisher Scientific) for 12–14 days, with the medium replenished every 3–4 days. Adipogenic differentiation and the presence of lipids in the cells was evaluated by staining with LipidTOX .For adipogenic differentiation, AdMSC were seeded at 1 × 103 cells cm−2 and cultured in MesenPRO RS Medium. After 3 days, culture medium was replaced with StemPro Osteogenesis Differentiation medium (ThermoFisher Scientific) for 21 days, with the medium replenished every 3–4 days. Osteogenic differentiation was evaluated by staining with Alizarin Red S (ACROS Organics). Quantitative evaluation of the stain has been performed by dye extraction using an aqueous solution of 20% (v/v) methanol  and 10% (v/v) acetic acid . The absorbance was measured at 450 nm wavelength.For osteogenic differentiation, AdMSC were seeded at 5 × 107 cells mL−1) in the center of a well of a 48‐well plate. After 2 h incubation at 37 °C in 5% CO2, chondrogenesis medium  was added to the well for 14 days, with the medium replenished every 2–3 days. Chondrogenic differentiation was evaluated by staining with Alcian Blue dye (Sigma Aldrich).For chondrogenic differentiation, micromass culture was performed by seeding 5 µL droplets of a high concentration cell suspension  for 5 min each. The samples were immersed in hexamethyldisilazane (Sigma Aldrich) for 2 min and were left to dry at room temperature in a desiccator. Samples were sputter coated with gold (Polaron E5000) and imaged using a Hitachi S3400N SEM at 5 keV.p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). Results are expressed as mean ± standard error of the mean  or as mean ± STDEV.Data were subjected to one‐way or two‐way ANOVA followed by Tukey’s tests for multiple comparisons between pairs of means, using commercially available software (GraphPad Prism). Statistically significant difference between experimental results was indicated by The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Five developmental trajectories of partnering in the ages 18–35 were identified by Vergunst et al. (2020). In my discussion of these trajectories, I included findings of three studies using the same dataset as Vergunst et al. First I showed that formation and maintenance of intimate relationships have different childhood precursors. It also became clear that family adversity and high inattention in childhood are systematic predictors of problems in investing in age‐graded roles in adulthood: in educational attainment, partnering, and becoming economically self‐reliant. A limitation of the study Vergunst et al. is the absence of adolescence. Inclusion of data on adolescent development could have clarified why effects of family adversity and childhood traits are very small and provided evidence for the role of parent–adolescent relationships. Finally, the study by Vergunst et al. demonstrates intergenerational transmission of problems across various developmental domains. This intergenerational continuity of vulnerabilities suggests developmental selection. Five developmental trajectories of partnering in the ages 18–35 were identified by Vergunst et al. : early‐pThe study by Vergunst et al. has multiple strengths: (a) the use of multi‐informant data, childhood behaviors were rated by teachers whereas partnership data at the ages 18–35 were derived from tax return records; (b) adequate controls of background characteristics (gender of child and family socio‐economic status), and (c) a big sample size allowing to assess adequately heterogeneity of partnering trajectories. The report also adds to the growing but relatively small number of studies demonstrate that childhood behaviors are predictive of adult behavior  adulthood such as finishing an education and becoming economically self‐reliant? A series of studies using the same dataset  to uncover temporal order between various developmental processes. The second approach delves deeper into the developmental process Meeus, . Whereas


The usage of preprint servers in ecology and evolution is increasing, allowing research to be rapidly disseminated and available through open access at no cost. Early Career Researchers (ECRs) often have limited experience with the peer review process, which can be challenging when trying to build publication records and demonstrate research ability for funding opportunities, scholarships, grants, or faculty positions. ECRs face different challenges relative to researchers with permanent positions and established research programs. These challenges might also vary according to institution size and country, which are factors associated with the availability of funding for open access journals. We predicted that the career stage and institution size impact the relative usage of preprint servers among researchers in ecology and evolution. Using data collected from 500 articles , we showed that ECRs generated more preprints relative to non‐ECRs, for both first and last authors. We speculate that this pattern is reflective of the advantages of quick and open access research that is disproportionately beneficial to ECRs. There is also a marginal association between first author, institution size, and preprint usage, whereby the number of preprints tends to increase with institution size for ECRs. The United States and United Kingdom contributed the greatest number of preprints by ECRs, whereas non‐Western countries contributed relatively fewer preprints. This empirical evidence that preprint usage varies with the career stage, institution size, and country helps to identify barriers surrounding large‐scale adoption of preprinting in ecology and evolution. We predicted that the career stage and institution size would impact the relative usage of preprint servers among researchers in ecology and evolution. Using data collected from 500 articles, we demonstrate that ECRs generate more preprints relative to non‐ECRs, for both first and last authors. We speculate that this pattern is reflective of the advantages of quick and open access research that is disproportionately beneficial to ECRs. The first 100 full‐text English articles from each journal were collected to produce a final dataset of 200 open access articles, 200 closed access articles, and 100 preprints. Fewer than 100 articles from Proceedings of the Royal Society B in 2019 fit our criteria, so the search was expanded to include publications in 2020 to achieve the desired sample size. The title of the article, number of authors, names of first and last authors, and author affiliations were collected. The total numbers of publications for first and last authors were determined using authors' Google Scholar profiles. Articles were excluded if the first or last author did not have a Google Scholar profile. By excluding the work of authors that do not have a Google Scholar profile, certain career stages may have been disproportionately excluded depending on the relationship between career stage and Google Scholar profile usage. Removing data disproportionately from certain career stages could have resulted in null or unclear relationships. However, we felt that our sample sizes across career stages were sufficient. The bioRxiv database was used to determine the number of preprints an author has submitted. We defined ECR as a student at any stage  to determine if the mean number of preprints differed by the country associated with the first author. We then used ArcGIS Pro (version 2.6.3) to visualize the distribution of preprints and proportion of early career researchers by country. The percent of total preprints represents the sum of first author preprints in each country divided by the total number of preprints counted in this study. The proportion ECR represents the proportion of preprints in each country with an ECR as the first author. A value of 1 indicates all preprints were submitted by an ECR and a value of 0 indicates that all preprints were submitted by a non‐ECR.3Proceedings of the Royal Society B = 100 , PLoS ONE = 100, Ecology = 100; Ecology and Evolution = 100, bioRxiv = 100). The quasi‐GLM indicated that career status of both first and last authors had significant associations with the number of preprints generated by the first author  or last author . When considering the totality of preprints generated by first and last authors from the 500 articles (n = 734), the United States and the United Kingdom contributed the greatest number of preprints by early career researchers, whereas non‐Western countries contributed fewer preprints .Spearman's rank correlation tests indicated no correlation between the number of preprints and total number of publications for the first author ; Data curation (lead); Formal analysis (lead); Investigation ; Methodology (lead); Project administration (lead); Visualization (lead); Writing‐original draft ; Writing‐review & editing . Layla MacKay: Conceptualization ; Data curation ; Formal analysis ; Investigation ; Methodology ; Writing‐original draft ; Writing‐review & editing . Sarah E. Haworth: Conceptualization ; Data curation ; Formal analysis ; Investigation ; Methodology ; Writing‐original draft ; Writing‐review & editing . Marie‐Laurence Cossette: Conceptualization ; Data curation ; Formal analysis ; Investigation ; Methodology ; Writing‐original draft ; Writing‐review & editing . Morgan N. Dedato: Conceptualization ; Data curation ; Formal analysis ; Investigation ; Methodology ; Writing‐original draft ; Writing‐review & editing . Kiana B. Young: Conceptualization ; Data curation ; Formal analysis ; Investigation ; Methodology ; Visualization (lead); Writing‐original draft ; Writing‐review & editing . Colin I. Elliott: Conceptualization ; Data curation ; Formal analysis ; Investigation ; Methodology ; Writing‐original draft ; Writing‐review & editing . Rebekah A. Oomen: Formal analysis (supporting); Investigation ; Methodology (supporting); Supervision (lead); Visualization (supporting); Writing‐review & editing .


Conventional synthetic vascular grafts require ongoing anticoagulation, and autologous venous grafts are often not available in elderly patients. This review highlights the development of bioartificial vessels replacing brain‐dead donor‐ or animal‐deriving vessels with ongoing immune reactivity. The vision for such bio‐hybrids exists in a combination of biodegradable scaffolds and seeding with immune‐neutral cells, and here different cells sources such as autologous progenitor cells or stem cells are relevant. This kind of in situ tissue engineering depends on a suitable bioreactor system with elaborate monitoring systems, three‐dimensional (3D) visualization and a potential of cell conditioning into the direction of the targeted vascular cell phenotype. Necessary bioreactor tools for dynamic and pulsatile cultivation are described. In addition, a concept for design of vasa vasorum is outlined, that is needed for sustainable nutrition of the wall structure in large caliber vessels. For scaffold design and cell adhesion additives, different materials and technologies are discussed. 3D printing is introduced as a relatively new field with promising prospects, for example, to create complex geometries or micro‐structured surfaces for optimal cell adhesion and ingrowth in a standardized and custom designed procedure. Summarizing, a bio‐hybrid vascular prosthesis from a controlled biotechnological process is thus coming more and more into view. It has the potential to withstand strict approval requirements applied for advanced therapy medicinal products. Substitution of blood vessels is required when the vasculature is affected by severe atherosclerosis or even stenosis. Besides synthetic vascular grafts with the necessity of ongoing anti‐coagulative treatment, autologous venous substitutes are often not available in the elderly, and allogeneic vascular grafts from brain‐dead donors require con‐comitant immunosuppression. Up to now, here the use of autologous vessels represents the gold standard for vessels with a diameter <6 mm, but bio‐artificial alternatives for narrow‐caliber vessels are urgently needed. Such bioartificial grafts ‐ manufactured with autologous cells and a suitable vascular prosthesis material are being developed. The prospective approach is to bypass diseased vessels with an up to three‐cell‐layer‐containing tubular scaffold as wall structure . In thisThe principle of developing a tissue‐engineered vascular graft (TEVG) includes several steps: suitable autologous vascular cells such as endothelial progenitor cells or vascular smooth muscle‐outgrowth cells could be isolated, for example, from a patient's peripheral blood. Alternatively, inducible pluripotent stem cells (iPS) or allogenic adult stem cells out of bone marrow or liposections or other niches of the human body are likely to be prepared and consequently differentiated to vascular cells. Human iPS are currently being explored for this application , since iThis review highlights the biotechnical issues and clarifies, what are the necessary prerequisites to cultivate a vascular graft as replacement vessels for clinical use. In an elderly society, there is a huge need of new bioartificial vessels, since patients often do not have enough autologous vascular substitutes. The article discusses different scaffold and hydrogel materials, cell sources with their risks and advantages as well as bioreactor technologies including suitable cultivation monitoring tools. The significance of the extra‐corporal cultivation process for such grafts is explained, which is the basis for physiological vascular maturation. The importance of 3D printing for vascular scaffold design is outlined. Among the diverse concepts, bio‐hybrid vascular structures cultured in a perfusion bioreactor appear as a realistic possibility for near clinical use.22.1Since xenogeneic cells impose a risk of inter‐species rejection, firstly human allogenic cells were tested for TEVGs. The problem of extra‐corporal expansion of these cells was remarkably solved by Poh et al., who developed a method to extend the lifespan of human endothelial cells isolated from senior donors. The group used genetic manipulation to achieve increased telomerase expression and growth . Lawson 2.2In the last 5 years, new aspects have emerged in the field of developing new (bio‐) hybrid vascular prostheses. Concepts that have proven themselves to the point of use in patients now often rely on bioresorbable synthetic scaffolds. In the past, Niklasson and Lange have already set an important model approach for new developments in this field with a synthetic scaffold for a small caliber poly(glycolic acid) (PGA) vessel with bovine cells at that time . Referri2.3The principle of an extracellular cultivation period inside a suitable bioreactor can be referred to as “in situ tissue engineering.” This concept was followed in parallel to approaches suggesting the use of a so‐called “living bioreactor,” that means the spontaneous implantation of the artificial vascular graft into a living organism for further seeding and integration into the vascular system of the recipient. As an example for this living bioreactor, Dahan et al. could successfully implant vascular grafts directly into an animal model, where spontaneous colonization with endothelial cells occurred in vivo . Which cTo optimize the seeded surface of the scaffolds and avoid thromboembolic complications of these artificial vessels, various surface modifications for the synthetic materials have been tested and the principle of dynamic cultivation of colonized scaffolds prior to implantation was found to be advantageous . Luo et 2.4A synthetic tubular scaffold and non‐immunogenic endothelial and vascular smooth‐muscle (‐like) cells can thus be marked as essential prerequisites for a standardized generation of bio‐hybrid vascular prostheses. Such bio‐hybrid vessels should undergo dynamic cultivation and conditioning in an intelligent bioreactor, and this total concept represents an attractive approach with high potential to reach clinical usability.3The design of an intelligent bioreactor system must feature various sensory elements, a robust system setup and the possibility to reuse the bioreactor containment for several cultivation processes of TEVGs. Different kinds of TEVG bioreactor setups were presented over the past decades, with perfusion bioreactors as the predominantly‐used model , 19, 20 Different papers emphasize such specific important bioreactor features. For example, the bioreactor of Huang et al.  offers tAnother comprehensive cultivation process for TEVGs  has been elaborated and improved over several years at the Leibniz University of Hannover in , 23, 24.The central element of the setup is the bioreactor chamber constructed out of polyetherimide, a high performance plastic material resistant to temperatures above 130°C, therefore suitable for heat sterilization. Furthermore, the material allows for the application of ultrasound imaging. The bioreactor has a tubular shape with an inner diameter of 3 cm. Depending on the required length of the implant, different lengths of the bioreactor up to 14 cm are possible. The tubular scaffold seeded with autologous cells is fastened centrally between the two end covers of the bioreactor chamber. Each end cover has two connectors for liquid medium supply. The diameter and lengths of the connectors vary with regard to the scaffold dimensions. The inner medium circulation (red) leads through the vascular graft and simulates the flow of blood. The outer circulation (orange) is used to fill the space surrounding the graft with liquid medium, simulating the interstitial space.The bioreactor is mounted on a motorized cradle driven by a stepper motor to rotate the graft ± 180 ° and avoid twisting the connected flexible tubes. Combining the characteristics of rotational and perfusion bioreactors into one system results in (i) enhanced mass transfer not only at the construct periphery but also within the internal pores of the tissue and (ii) prevention of gravitational effects on the graft which might lead to asymmetric cell growth during cultivation.3.1To ensure the quality of a TEVG, an automated monitoring and control system is necessary. The system presented in Figure 2. This is due to the formation of a local “lubrication chamber” in the flow process of the blood [2 and over several days was successfully established in suitable shear stress mini‐compartments, leading to enhanced expression or activation of cell differentiation markers. Pressure here was applied intermittently or uniformly to support the maturation of differentiated endothelial cells , or SMCs  [Pressure conditions as in the human circulation  represent a differentiation signal for maturing vascular cells. However, due to the blood flow and regarding the Hagen‐Poiseuille flow laws applicable to physiological conditions, pressures significantly lowered compared to the system pressure act on the inner skin of the vessels in the range of maximum 50 dynes/cmhe blood . Shear sy chain) , 27. In y chain) . Besidesy chain) .2 on the inner wall of the cultivated TEVG, which is required for a optimal cell adhesion to the scaffold. The baseline pressure is altered by applying pressure on the medium in the bulk medium container of the internal circulation. A non‐invasive ultrasonic flow sensor can be placed upstream of the vascular graft to assess the flow rate and to detect air bubbles. The rotating bioreactor chamber, bulk medium container, tubing, and all attached actuators and sensors are placed inside an incubator to provide temperature and humidity conditions appropriate for the cultivation process. Electronics located inside the incubator are sealed against the harsh conditions. All electrical components placed inside the incubator are specifically designed to minimize power dissipation to reduce the risk of overheating the well‐insulated incubator and the components placed inside.In the system presented above Figure , the phy3.2To realize a close cultivation control, the exemplarily presented bioreactor system includes enzyme‐based electrochemical biosensors to monitor the glucose and lactate concentration of the media . These dAmbient oxygen and carbon dioxide concentrations can be controlled and monitored by the incubator. Gas‐permeable tubing allows gas exchange with the medium. Physiological conditions for either one of the circulation cycles can be independently controlled by deploying a separate gas inlet for the conditioning of the medium and non‐permeable tubing for the respective medium circulation.2) and carbon dioxide (pCO2) in the medium can be controlled online by fluorescent analyte‐sensitive sensor spots. Since only small changes of these parameters are expected, a low frequency sampling rate is needed and the sensors can be located outside the medium circulation as part of the established FIA. This also results in decreased costs of these disposables since both medium circulations can be assessed with only one (non‐sterile) sensor for each parameter. All sensors that are part of the FIA system can be used to monitor multiple bioreactor setups, thus decreasing overall cost and setup time.pH and partial pressures of both oxygen  are therefore obtained already before the end of the cultivation period. The provided geometric structure information also facilitates fluid dynamic simulations to further evaluate the mechanical characteristics of the vessel .Other methods for 3D visualization, that contain a higher visual resolution exist in different formats: a Micro‐CT with a possibly cell deteriorating side effects of X‐rays , magneti3.42 ( = quasi‐static conditions).To demonstrate the suitability of the bioreactor system depicted in Figure 2 are still ahead, and in this context, others have successfully cultured fibrin‐decorated xenogeneic scaffolds with SMCs under a flow rate of up to 10 ml/min, and here a burst pressure of up to 200 mm Hg could be reached [Fluorescence staining revealed a confluent layer of endothelial cells on the inner surface of the vessel, which is crucial for subsequent vascular functionality. Dynamic cultivations with up to 20 dynes/per cm reached .44.1Xenogeneic and allogeneic scaffolds were tested as possible natural scaffold materials. Decellularization concepts aim to avoid immunogenic reactions but also residual concentrations of cell‐toxic detergents in the recipient, and here an alternative concept for decellularization based upon caustic soda was realized by Eyre, Haverich et al. . Dahan  produced4.2The review by Cordelle  describeCollagen is the most used natural polymer in vascular tissue engineering. As the main component of native extracellular matrix (ECM), it comprises properties such as low antigenicity, high biocompatibility, biodegradability, and is adhesive , 42. DueThe more elastic fibrin gels (using fibrin formed out of fibrinogen after adding thrombin) are of benefit in vascular tissue engineering, since (i) it can be produced with the patient's own blood and (ii) it binds to critical proteins that direct cell fate, such as fibronectin and vascular epithelial growth factor (VEGF). One disadvantage could be that fibrin may interact with local coagulation cascades. Fibrin is mostly used as bio‐ink together with more stable scaffold materials , but wasChitosan (CTS) is derived from chitin through its alkaline deacetylation, acts anti‐inflammatory and anti‐coagulative  and has Elastin is a natural elastic polymer that occurs in the natural wall of arteries. Nguyen et al tested a combination of elastin with collagen in seeding experiments with regard to a potential vascular graft. The component elastin had an advantageous effect on ECM organization and contractility of seeded SMCs , and vasAltogether, the natural materials presented here have excellent molecular properties for optimal cell growth, and some of them act as anti‐thrombotic agents. However, they are mostly used – except of silk – as decorating and cell adhesive material together with other, more stable materials in vascular tissue engineering, and were not consequently followed as single‐component‐vascular graft for patients up to now.4.3Scaffolds can also be produced out of biodegradable polymers. Here the idea is, that in parallel to the degradation process of the polymer, the seeded cells overtake the regimen and produce natural extracellular matrix and finally form a tissue layer. Currently, many different materials are available that can be considered as scaffold materials for a vascular prosthesis´ scaffold. PGA as an established polymer has a high melting point >200 °C, and high tensile strength (12.5 GPa) and was tested as vascular scaffold already 20 years ago , but exh4.4Of note, PDO is a colorless semi‐crystalline polymer with a melting point of 180–190°C. PDO ‐ considered to have a fast to moderate degradation time (∼3–6 months) ‐ is not very deformable, but flexible and is well established as a suture material for 30 years as monofilament PDS. PDO has already been tested in combination with adipogenous tissue derived mesenchymal stem cells (AD‐MSCs)  and suppAs depicted in the schematic of Figure 4.5The method of electrospinning nanofibers on a cylinder has also long been explored for development of tubular vascular scaffolds. Electrospun tubes were wrapped in a thicker polymer coil in order to improve the mechanical properties of these vascular scaffolds. Constructs seeded for example with MSCs were tested in a rabbit model and showed long patency throughout implantation . The dis4.63D printing represents a new approach for matrix and scaffold fabrication and belongs to the class of additive manufacturing processes in which, in contrast to the classic subtractive processes , the desired object is produced by applying material layer by layer. Numerous different printing processes have developed from the various fields of application, the variety of materials processed and other general conditions such as requirements for accuracy or surface finish of the manufactured parts. Regardless of the 3D printing process, the workflow generally comprises three main steps: (i) The desired geometry is created with the help of computer‐aided processes, usually using CAD software before (ii) being processed by specialized slicer software, which breaks down the geometry into individual layers (“slicing”) and generates printer‐specific commands from each layer. The command set is processed by the 3D printer, and (iii) the desired geometry is manufactured by selective material deposition . 3D prinPorosity generated by 3D printing with significantly smaller pore diameter of scaffolds supports cell adhesion and can be even more enhanced by staggering the layers and using lattice patterns as well as hexagonal and gyroid patterns. Polymers used in the context of 3D printing can be processed by digital light printing (DLP). One example is poly (propylene fumarate) (PPF), a biodegradable polyester. This compound is a UV resin, solved in diethyl fumarate, and is activatable via bisacrylphosphrine oxide as photoinitiator for crosslinking . PPF‐scaSummarizing, 3D printing offers the benefit of producing scaffolds fast and reproducible with a highly defined structure. The challenge for 3D printing with regard to vascular grafts is to produce a seamless tubular structure with high mechanical elasticity. The following Figure Future developments in 3D printing must also comprise concepts for printing multilayered vascular wall structures, suitable as blood vessel scaffolds. This and the delicacy of the resulting process vote for an exploration of the method of Melt Electro‐Spinning and Writing (MESW) as an attractive new method in 3D printing. The Melt Electro‐Writing (MEW) process is attracting special attention in the field of bioprinting, as this method allows very thin fibers with a fiber diameter of typically 2–50 μm that can be deposited in defined structures .55.1As mentioned earlier, not only the antithrombotic surface design of suitable scaffolds, but also mechanical resilience is an important issue in a vascular graft. This led to a number of experimental approaches in which, in addition to surface modifications, different cell sources were used for colonization and even multicellular wall structures could be created.Heine and Haverich  describe2) on silk‐fibroin‐modified poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate (SF‐PHBHHx), a confluent structure developed in the form of lamellae [Xenogeneic cell isolation protocols were successfully explored  but as dlamellae .The problem with autologous cells is that they are needed in high numbers for endothelialization of suitable scaffolds. A cell isolation method for obtaining endothelial progenitor cells (EPCs) from peripheral blood was earlier established and recently optimized.Mononuclear cells are selected during cultivation and characterized as endothelial cell forming cells (ECFCs) as a subset of EPCs by flow‐cytometric analyses  These ceAs an alternative solution to the concept of isolating autologous cells with ex vivo expansion, human leucocyte antigen (HLA)‐type‐matched human inducible pluripotent stem cells (hiPS) may also be used. In parallel, HLA‐gene edited hiPS with reduced HLA‐molecules I and II expression are being developed . These i5.2For conditioning of either endothelial or SMCs, dynamic cultivation in a suitable bioreactor is important and supports the differentiation process of immature progenitor cells into mature vascular cells. It was for example beneficial for the differentiation of mesenchymal stem cells to smooth‐muscle‐cell‐like cells on suitable biodegradable scaffolds (made of PCL/PLA). Dynamic cultivation here may comprise not only laminar flow but also pulsatile flow conditioning, and may thus have anti‐thrombogenic and anti‐inflammatory effects promoting the generation of a vascular graft not endangered by fast thrombogenicity after implantation . How deeWe report an approach for dynamic cell culturing for ECFCs. ECFCs exhibit significant changes in the axial ratio after both laminar (LC) and pulsatile (PC) shear stress cultivation, whereas for endothelial cells from a cell line such as HUVECs a significant change is only observed after pulsatile flow Figure . During 6Bio‐artificial grafts, that have already reached the level of clinical trials, were mainly composed of immune‐neutral components to prevent thrombosis. Some of them showed a mechanical weakness and developed stenosis. Cell conditioning and graft maturation in a bioreactor therefore appears advantageous.Suitable bioreactor systems, equipped with multi‐modular functions, are the basis for the successful production process of a vascular graft and seem in tangible proximity. A bioreactor system can act as a test setup providing physiological conditions, thus simulating the human body during vasculogenesis. Various harsh conditions for electronic elements have to be considered, and corresponding suitable solutions have to be realized. Dynamic cultivation is able to produce mechanically stable endothelial monolayers on the synthetic scaffold and trains the cells to expose an anti‐thrombogenic character. Ultrasound monitoring and 3D visualization of the growing graft in the bioreactor containment is practical, but has a comparably low image resolution. In contrast, alternative methods such as OCT, MMP, and SHG with high resolution have a minor penetration depth and may only allow surface studies of the vascular grafts and are not yet integrable in bioreactor systems due to their enormous apparative expenditure up to now.Among the diverse materials used for vascular tissue engineering, natural gelatinous materials such as fibrin, chitosan, and others have not yet found their way as scaffold‐free vessels resilient enough for clinical use. Silk by contrast is a very attractive material especially for manufacturing with electrospinning. There are many suitable, biocompatible materials such as biodegradable polymers that were tested with cells so far. Here exists the vision, that cell matrix production will overtake the mechanical strength of the earlier scaffold. For their successful use as part of a vascular graft, degradation properties will be of major importance. Some of these polymers were processed as electrospun vessels, and most of them were decorated with adhesive additives for optimal cell growth and increased anti‐thrombogenic or antimicrobial properties. A new trend is to generate vascular 3D printed tubes with microstructured surface or as macroporous scaffolds for capillary structures. Among the huge number of varying polymers tested so far, PCL as outer support, PDO as inner layer with its medium degradation time and human fibrin with adhesive properties may be a promising new combination of a natural and synthetic polymer to build up a vascular scaffold. Processing different biodegradable polymers by 3D printing in form combined extrusion printing and melt electrospinning writing represents a new attractive technology to generate such multi‐material pre‐designed, microstructured scaffolds. Of course, the resulting biomechanical stability must be extensively tested, but some 3D printed scaffolds have already shown a sufficient quality.Recruiting a cell number high enough for seeding vascular tissue engineered scaffolds, but with a low immunogenic profile, remains a problem to be solved. Xenogeneic and allogenic cell sources impose the risk of immunological identification and following fast immunological clearing reactions. In contrast and besides promising results concerning iPS, there are possibilities to isolate autologous endothelial progenitor cells e. g. out of blood and expand them. Furthermore, blood isolated smooth muscle outgrowth cells, which are differentiated to target cells in a distinct milieu, show great potential to be used as an autologous substitute for SMCs.Based on all these promising new approaches, standardized procedures for generation of vascular grafts are envisioned. Results should withstand durability tests to act as new and powerful replacement vessels especially for elderly or comorbid patients without alternatives. 3D printing here seems to be a highly sophisticated supporting technology in this context.The authors declare that there are no conflicts of interest.There were no ethical or legal concerns about the use of liposection material for isolation of adipose tissue derived human mesenchymal stem cells shown in some of the exemplary results presented here. Liposection material was obtained after operations in the Dept. of Plastic and Reconstructive Surgery of the Medical School Hannover (MHH), and this was audited on behalf of the ethics committee of the MHH (reference number 3475‐2017).


The COVID‐19 pandemic has strongly disrupted academic activities, particularly in disciplines with a strong empirical component among other reasons by limiting our mobility. It is thus essential to assess emergency remote teaching plans by surveying learners’ opinions and perceptions during these unusual circumstances. To achieve this aim, we conducted a survey during the spring semester of 2021 in an environmental science program to ascertain learners’ perceptions on online and onsite learning activities in ecology‐based modules. We were particularly interested not only in comparing the performance of these two types of activities but also in understanding the role played by learners’ perceptions about nature in shaping this pattern. Environmental science programs are rather heterogeneous from a conceptual point of view and, thus, learners may also be more diverse than in traditional ecology programs, which may affect their interest for ecology‐based modules. We assessed connectedness to nature by computing the reduced version of the Nature Relatedness Scale. Here, we found that online activities systematically obtained significantly lower scores than onsite activities regardless of the wording employed, and that altruistic behaviors were prevalent among learners. Interestingly, scores for both onsite and online activities were strongly influenced by learners’ connectedness to nature, as learners with a stronger connection to nature gave higher scores to both types of activities. Our results suggest that an effort to improve the efficacy of remote learning activities should be the focus of research about teaching methodologies in predominantly empirical scientific disciplines. We found that online activities systematically obtained significantly lower scores than onsite activities regardless of the wording employed, and that altruistic behaviors were prevalent among learners. Interestingly, scores for both onsite and online activities were strongly influenced by learners’ connectedness to nature, as learners with a stronger connection to nature gave higher scores to both activity types. The COVID‐19 pandemic is representing a huge challenge for learning and teaching, not only by limiting our mobility  is a standard questionnaire aiming to quantify connectedness to nature  Are online and onsite learning activities well connected to key concepts in ecology? (2) What is the perception of undergraduates on online and onsite learning activities? (3) How undergraduates perceived empirical and altruistic learning activities? (4) Was connectedness to nature (NRS) an optimal predictor of these perceptions?22.1N = 22), year‐3 Aquatic and Urban Ecology (N = 16) and year‐4 Ecology in a Changing World (N = 11). All undergraduates but one from South Korea were Chinese nationals. We obtained ethics approval from XJTLU's University Ethics Committee (UEC) through the Educational Development Unit (EDU) and informed consent from the undergraduates before conducting the survey. Across March and early April, undergrads were engaged in different types of online and onsite activities, such as terrestrial and aquatic biodiversity monitoring, checking nest boxes, and observing recordings of animal behavior. They also experienced a fully online learning mode during the second semester of the previous academic year, so that all undergraduates were able to express informed opinions about both types of learning approaches. Most of these field activities were instrumental for undergraduates to complete their coursework, yet other activities represented an altruistic contribution to the class—any undergraduate was able to make use of the collected data.In this study, we conducted a survey among 49 undergraduate students between 19 and 23 years old enrolled in the Environmental Science program of the Xi'an Jiaotong‐Liverpool University (XJTLU)  during the second semester of the academic year 2020–2021. Undergraduates were enrolled in different ecology modules, namely year‐2 Aquatic Field Skills . We did so by performing pairwise t‐tests, a standard repeated‐measures approach, so each pair of answers is compared for each interviewee. We computed the average for questions about online and onsite learning separately and performed a pairwise t‐test to see if any patterns were consistent when considering all the questions together. Connectedness to nature (NRS) was calculated as the average of the NR‐6 questions for each interviewee on the assumption that higher scores would imply stronger connectedness to nature and vice versa.nlme  model fitted with restricted maximum likelihood using the average from questions about online learning as dependent variable and the average from the NR‐6 questionnaire as independent variable. We repeated this procedure alternatively using the average from questions about onsite learning and the scores of questions Q15 and Q16 about altruistic behaviors as dependent variables. We included module  as random factor. LME models were constructed using the package 3In all pairs of questions, undergraduates gave higher scores to onsite than to online learning Table  and 2. MFurthermore, undergraduates were keen to share collected data with other undergraduates Table  and 2e aβ ± SE = 0.30 ± 0.13, t3,45 = 2.24, p = .03), and with average scores of questions about online learning    Figure .4The current pandemic has strongly disrupted academic activities ; Data curation ; Formal analysis ; Methodology ; Writing – original draft (lead). Mingxiao Yan: Conceptualization ; Data curation ; Formal analysis ; Writing – review & editing . Yixuan Hong: Conceptualization ; Data curation ; Formal analysis ; Writing – review & editing . Yu Zeng: Conceptualization ; Data curation ; Formal analysis ; Writing – review & editing . Sihao Chen: Conceptualization ; Data curation ; Formal analysis ; Writing – review & editing . Peng Zhao: Conceptualization ; Data curation ; Formal analysis ; Writing – review & editing . Yi Zou: Conceptualization ; Data curation ; Formal analysis ; Writing – review & editing .