Hydrogels have progressed from single-network materials with low mechanical integrity todouble-network hydrogels (DNHGs) with tough, tunable properties. Inthis work, we introduce a nanocomposite structure into the first networkof a DNHG. Amine-functionalized silica nanoparticles (ASNPs) werecovalently cross-linked by forming amide bonds through the carboxylicgroups of polyacrylic acid (PAAc) in the first network. DNHGs withvarying sizes of ASNPs  and varying concentrations were explored and compared to a controlwithout a nanocomposite structure. Compressive strengths improvedfrom 0.10 MPa for the control to a maximum of 1.28 MPa for the PAAc/PAAmDNHGs. All hydrogels experienced increased resistance to strain witha maximum of 74% compared to 45% for the control. SEM images of freeze-driedgels showed that ASNPs were integrated into the gel mesh. Nanoparticleretention was calculated using thermal gravimetric analysis (TGA)with improved retention values for larger ASNPs. New DNHG compositeshave been formed with improved mechanical properties and a potentialuse in tissue engineering and biomaterial applications. The mechanical properties of hydrogelsremain to be a challenge for these applications. Conventional hydrogels,which are composed of single hydrophilic polymer networks, are usuallysoft, brittle, and weak, limiting their progression and success inclinical applications.3 They often havemechanical and tensile properties that are sub-megapascal with strainsof less than 100%. Due to their highly hydrated states and low polymerdensity relative to their water content, hydrogels exhibit low mechanicalstrength and weak structural integrity. Another reason for their lowmechanical properties is the heterogeneity of the polymer networkstructure that is formed during gelation.5 Hydrogels withheterogeneous polymer networks fail at low forces as the force appliedconcentrates around the shortest chains. Therefore, several new typesof hydrogels have been developed to improve the mechanical performanceof hydrogels by dispersing the applied load within the microstructureto reduce crack propagation. These new hydrogels include double-networkhydrogels (DNHGs),8 photoactivehydrogels,11 nanocomposite hydrogels,16 slide-ring gels,19 and supramolecular polymernetwork gels20 among several other uniquehydrogels.24Hydrogels are soft and wet materials thatare made up of cross-linkedpolymeric macromolecules and water. They have the ability to holdup to more than 90% water while maintaining their structural integrity.The characteristic similarities between hydrogels and soft tissueshave drawn attention to this class of material as biomaterials. Theviscoelastic polymer networks of hydrogels resemble the network structureof the extracellular matrix (ECM) found in biological tissue thatis composed of more than 60% water. Therefore, a lot of focus hasshifted toward developing hydrogel-based biomaterials to mimic nativehuman tissue and facilitate tissue repair, replacement, and regeneration.For example, load-bearing tissue such as hyaline cartilage, whichis made of approximately 75% water, exhibits a compressive fracturestress of 36 MPa6 They have shown theability to hold high water content while maintaining mechanical strengthand toughness. The first network normally consists of a tightly cross-linkedrigid polyelectrolyte polymer, while the second network is made ofa sparsely cross-linked neutral polymer, typically acrylamide.25 Gong et al.’s originalDNHG achieved exceptional mechanical properties with a fracture stressof 20 MPa and Young’s Modulus of 0.3 MPa with water contentover 90%.6Of those examples, DNHGs have made the largest strides intermsof improving mechanical properties. DNHGs consist of two separateand contrasting polymeric networks that result in a synergistic effectof the two polymers.13 copper nanopowder,26 laponite clay,15 nanoceria,27 and nanocellulose crystals.29 Nanoparticles enable unique properties in the hydrogels, includinginternal physical reinforcement to external strain, enhanced topographyfor cellular attachment, conductivity, bacterial resistance, antioxidation,magnetic responsiveness, and a potential for electrical signals andsensing.32 The addition of nanoparticles with functional chemical groups ontheir surface can result in covalent or ionic cross-links with thepolymer network of the hydrogel or adsorption of the polymer chainsby being entrapped within the hydrogel network.DNHGs have progressedmore with the introduction of nanocompositestructures that provide unique microstructures that further enhancethe mechanical properties of the hydrogels. Nanocomposite hydrogelsconsist of nanoparticles that are integrated into the polymer networksduring hydrogel synthesis in water. Several types of nanoparticleshave been used in nanocomposite gels, such as silica nanoparticles(SNPs),33 These includeadhesives for wound healing,30 implantablebio-receptive scaffolds,32 flexible conductivesensors, and bio-wearables.33 For example,in tissue engineering, the application of hydrogels is often limiteddue to different factors including poor mechanical and limited cellattachment sites,34 and optical properties.35 However, the addition of nanocomposites addressesthese limitations by enhancing the mechanical properties,3 which provides cellular adhesion sites for improvedbio-receptivity,36 delivery of growth factors tosupport cell growth,36 increased thermalstability,37 and self-healing propertiesand promotes stem cell differentiation.32 Further, conventional hydrogels used for drug-delivery systems oftenrelease drugs in an uncontrolled and unpredictable manner.38 Nanocomposite structures provide control overthe cross-linking density, microporosity, stimulus responsiveness,and mechanical properties of the hydrogel, providing more accuratelong-term control for drug release.38These improvementsin the physical and biochemical as well as themechanical properties of hydrogels through the addition of nanocompositestructures have found use in a wide range of biomedical and tissueengineering applications.41 To help evade these problems, it is important thatnanoparticle and polymer interactions are robust. This can be achievedby functionalizing the surface of the nanoparticles with chemicalgroups that are able to directly create covalent links with the polymersin the hydrogel. In our previous studies, amine-functionalized nanoceria(ANC) were used as initiators for a redox surface-graft polymerization.A PAMPS first network was grafted on the surface of ANCs followedby a PAAm second network, resulting in enhanced mechanical propertiesof an ANC double-network hydrogel.12 However,this method requires a thermal process and constant sonication toinitiate the polymerization. Functional silica nanoparticles can insteadbe used at room temperature or through photopolymerization when asuitable initiating system is chosen, providing more versatility tothe synthesis technique of the gels.However, nanocomposite structures can also cause detrimental effectson hydrogels if they are not incorporated carefully: for an inhomogeneousdistribution, particle agglomeration can lead to stress concentrationsand the lack of covalent links between polymer strands and nanoparticlescan cause mechanical properties to be lower than the theoretical combinationof its parts.2 groups. Amine-functionalized silica nanoparticles (ASNPs)with varying diameters  and loading concentrations will be used to cross-link the PAAc firstnetwork. The objective was to use carbodiimide chemistry to form amidebonds between the carboxylic group of PAAc and the amine groups ofthe ASNPs. The hypothesis was that the first-network gel composedof ASNP-PAAc will subsequently be swollen and polymerized in a secondnetwork monomer solution to form a nanocomposite double-network hydrogel.In this work, acrylic acidwas chosen as the polyelectrolyte forthe first network and acrylamide was the neutral polymer for the secondnetwork. Polyacrylic acid is a versatile polymer that is commonlyused in both tissue engineering and industrial engineering. PAAc hasin its structure terminal carboxylic groups that can form covalentamide cross-links through carbodiimide chemistry through −NH4OH concentration. ASNPswere successfully synthesized by post-synthesis functionalizationusing APTES. ASNPs were dispersed in water and sonicated for sizeanalysis using DLS. O-acylisourea). NHS was used to providea more stable intermediate for reactions with primary amines fromthe ASNPs to form covalent amide bonds carbodiimide(EDC). PAAc was dissolved in MES buffer with EDC to form an unstablereactive ester . The water contentranged from 88–92%. All hydrogels reached a plateau at approximately150 h after being swollen in water. Previous research has shown thatan increasing nanoparticle cross-linking density in the first networkcould result in hydrogels exhibiting a decrease in swelling properties.46 Once the polymeric networks begin to swell,the hydrophobic groups are exposed. This results in hydrophobic boundwater known as “secondary bound water”.46 The “total bound water”, which is the collectivebound water of the primary and secondary states, is considered theinitial swelling state. Due to the osmotic driving forces in the hydrogels,the polymeric networks continue to absorb water until an equilibriumstate is reached. This equilibrium would be due to osmotic forcesbeing canceled out by retraction forces of the swollen polymer networks.This additional water is considered “bulk water”46 and is the plateau of water absorption. Figure S1 and Table S2.The loss of ASNPs during swelling suggests that cross-linking maynot have been complete for the first network or that excess ASNPsmay be floating unbound and were therefore washed-out during swelling.It is not possible to quantify the mass of nanoparticles that remainin the PAAc/PAAm hydrogels through FTIR. Therefore, ASNP retentionwas calculated using thermogravimetric analysis (TGA) on swollen hydrogels.FTIR results showed that unreacted AAm monomersand non-cross-linked PAAc were present in the system and were washedout after swelling in water, along with ASNPs. Further analysis canbe found in the 47 and thereforehave less functional groups available for cross-linking per unit massof ASNPs. The DTG curve for 10 wt % shows a shift to the right comparedto the control, meaning that higher temperatures and more energy arerequired to break the bonds and burn off the material. This suggeststhat the 10 wt % ASNP loading had improved cross-linking relativeto other concentrations. Swollen PAAc/PAAm hydrogels weretested under uniaxial mechanical compression, as shown in For 150 nm ASNPS, the highest compressive stress atfailure wasat 0.88 ± 0.04 MPa and 72 ± 4% with 10 wt % ASNPs. The nanocompositestructure in the hydrogels had an impact on the mechanical propertiesof the hydrogel. This was likely due to the bonds between PAAc andASNPs that held the polymer chains together while under stress, whichwere not present in control hydrogels. A trend canbe seen across all hydrogels where the 10 wt % loadingin the first network provided the best results for increasing strainand failure. In comparison, the 2.5 wt % loading was too low to providean impact on the mechanical properties, especially with sizes of 50and 100 nm. Both 20 and 40 wt % loadings potentially resulted in unboundor non-cross-linked ASNPs, which may result in agglomeration of particlesor non-cross-linked regions of the gel. ASNPs may agglomerate intoclusters and cause an inhomogeneous distribution within the gel, resultingin a lack of covalent links between polymers and nanoparticles. Thiscan result in reduced mechanical properties. This highlights thatthe desirable effects of ASNPs may be reached with lower concentrationsin the range of 10 wt %.Ultimately, the cross-linking chemistryof the first network isvital to the success of such tuning of the physical properties ofPAAc/PAAm hydrogels. The combination effect of the nanoparticle concentrationand size can impact the mechanical properties of the hydrogels. Thisis summarized and highlighted in 49 Such topography and anchorage points could be useful for cell proliferationand growth in tissue-engineering applications.Hydrogel samples were frozen at a −80 °Covernight and freeze-dried for two days before SEM imaging. In For the first time, novel nanocompositedouble-network hydrogelswere synthesized via carbodiimide coupling of amine-functionalizedsilica nanoparticles and polyacrylic acid as the first network. Thesecond network was formed using polyacrylamide through photopolymerization.The final PAAc/PAAm hydrogels with ASNPs showed tailorable mechanicalproperties through the integration of nanoparticles with varying sizesand loading concentrations. This study proved the importance of thenanoparticle size and loading concentration and their impact on swellingand mechanical properties. SEM images revealed that ASNPs were integratedinto the polymeric struts of the hydrogel mesh. The interaction ofpolymer chains with the ASNPs is believed to form an embedded micronetworkthat improves the mechanical properties of the hydrogels with a maximumcompressive stress of 1.28 MPa compared to 0.1 MPa of the control.The mechanical properties of these nanocomposite gels show an improvementto current nanocomposite IPN gels and are comparable to other nanocompositeDNHGs. Using different monomers, nanoparticles, and cross-linkingchemistry, we were able to achieve similar properties to our previouswork on ANC gels (1.78 MPa). The physical and chemical cross-linksprovide a key element for tailorability in these hydrogels and theirapplication. These improvements in properties have broad applicabilityin biomedical sciences and tissue engineering such as in adhesivesfor wound healing, drug-delivery agents, implantable scaffolds, flexiblesensors, and bio-wearables.2 groups on the surfaceof the ASNPs , equivalent to 2 g, was dissolved in 4 mL of MES buffersolution at pH 5.5. The 2.5 wt % ASNPs with a 50 nm diameter relativeto PAAc were dispersed in 1 mL of MES buffer and placed in a sonicationbath for 30 min until fully sonicated. EDC was added in the powderform to the PAAc solution at a mass ratio of 1:2.7 to ASNP, and NHSwas added at a mass ratio of 1:0.3 to EDC based on previous worksand systemic experimental trials.50Using carbodiimidechemistry, the tertiary −NHhe ASNPs 7 will fohe ASNPs 8. Succeshe ASNPs 7a,b.Fir2O. The hydrogels wereplaced in the AAm monomer solution and left until they were swollen.Once the hydrogels were swollen, they were placed in the UV cross-linkerfor photopolymerization of the second network to form the double-networkhydrogel. The UV cross-linker was set to 5400 s at maximum UV intensity(average 2900 μW/cm2) of 365 nm of light. This 5400s was found to be sufficient time for gelation. The double-networkhydrogel is then removed and dried at 60 °C before being immersedin water for 14 days. The process was repeated for 2.5, 10, 20, and40 wt % for 50, 100, and 150 nm ASNPs. A control DNHG was formed followingthe same procedure without ASNPs, EDC, and NHS. The synthesis routeis depicted in Next, the second network was formed using AAm. Anamount of 2.5g of AAm, 1 wt % (25 mg) Irgacure 2959, and 1 wt % (25 mg) BIS weredissolved in 5 mL of deionized HHydrogels with varying ASNP sizes and compositionswere dried at60 °C directly post synthesis for five days to reach a fullydry state. Subsequently, the dried hydrogels were ground into a powderand used for characterizing the chemical content post synthesis, referredto as “fresh” samples. Samples from each batch werealso placed in water directly post synthesis to be swollen for upto 14 days. Compression studies were conducted on the fully swollenhydrogels once the swelling study was completed. Swollen hydrogelsare referred to as “swollen” samples and were also usedfor TGA and FTIR analysis.


Alligator mississippiensis) locomoting on a treadmill. Pressure recordings documenting the cardiac, ventilatory, and barostatic influences on the CSF were in good agreement with previous studies. During locomotion the CSF exhibits sinusoidal patterns of pressure change that spanned a mean amplitude of 56 mm Hg, some 16 × the amplitude of the cardiac-linked pulsations. These sinusoidal CSF pulsations were closely linked to the locomotor kinematics, particularly the lateral oscillations of the alligator’s head. Data recorded from the freely moving alligators suggest that fluid inertia, body cavity pressures, and likely other factors all influence the CSF pressure. The clear relationship between movement and CSF pressure described in this study suggests that the paucity of studies examining human CSF dynamics during movement should be addressed.To examine the influence of movement on cerebrospinal fluid (CSF) dynamics, intracranial subdural pressure recordings were taken from sub-adult alligators ( In order to fulfill its diverse functional roles, the CSF must circulate; studies have postulated that abnormal CSF circulation may be the causal agent for a variety of neurological disorders, including Alzheimer's3 and syringomyelia4. Despite the importance of the CSF fluid dynamics, little is known about the magnitude, or even pattern, of "normal" CSF flow5. The CSF exhibits pressure pulsations corresponding to the cardiac cycle6; these pulsations have been thought to influence the dynamics of the CSF within the skull7, but this interpretation has been challenged8. Studies have shown that the ventilatory cycle generates significant levels of CSF flow9, primarily along the spinal cord10. Changes in the internal pressure of the body cavities, the intrathoracic and intraperitoneal pressures, can also influence the CSF dynamics11; it is unclear how these cavity pressure changes relate to the cardiac and ventilatory influences12.The cerebrospinal fluid (CSF) plays a key role in the development13. These different approaches for CSF data collection have two common features: 1) they provide clear records of cardiac and ventilatory patterns14, and they are largely incompatible with a moving subject15. Experimental stimulation of the myodural bridge, skeletal muscle fibers that insert onto the dura, demonstrated that muscle contraction can directly influence CSF pressure17. Recent work revealed that the undulatory movement of a snake's body, during fictive or conscious locomotion, resulted in CSF pressure pulsations of greater magnitude than those associated with either the cardiac or ventilatory cycles18.The emphasis on cardiac and ventilatory forces acting on the CSF reflects the underlying experimental evidence. Almost all of the data on CSF fluid dynamics were collected surgically on anesthetized animal or human subjects, or were recorded from human or animal subjects using magnetic resonance imaging (MRI)Alligator mississippiensis) employs a range of limb kinematics, with the main variable being the range of flexion in the limb. With high degrees of flexion at the shoulder/pelvis and elbow/knee, the proximal portion of the limb is held nearly horizontal; in this “push-up” position the ventral surface of the body is close to the substrate, so this is referred to as the “low walk.” With lower degrees of flexion at the shoulder/pelvis and elbow/knee, the proximal portion of the limb is held more vertical; in this semi-erect position the ventral surface of the body is further from the substrate, so this is referred to as the “high walk”. The low walk and high walk represent a functional continuum. Associated with this continuum is a range of lateral displacement of the limb  and corresponding lateral oscillation of the head, trunk, and tail base . Alligators have anatomical specializations to limit (but not eliminate) the undulatory displacement of the axial skeleton during locomotion19. Alligators are capable of shifting limb mechanics mid-stride and generally exhibit a wide range of limb angles20; because of this kinematic plasticity, the relationships among limb angle, body undulation, and locomotor velocity are complicated in Alligator and all crocodylians. By first training sub-adult alligators to locomote on a treadmill, it was possible to directly record intracranial CSF pressure from an unanesthetized vertebrate performing normal limb-based locomotion.The present study was undertaken to see if limbed locomotion has similar influence on CSF pressure as was shown with vertebral displacement in snakes. During terrestrial locomotion the American alligator (2). Power spectral analysis of the exhalatory CO2 data revealed a dominant frequency at 0.08 Hz  than the cardiac-linked pulsations recorded while the animal was under anesthesia. Power spectral analysis of the CSF pressures recorded when the animal was not being actively ventilated revealed simpler spectra , and a slight increase in the amplitude of the cardiac-linked CSF pulsations . In all of the locomotor trials, there was a close temporal congruence between the lateral oscillations of the alligator's head during locomotion, and the sinusoidal pattern of CSF pulsations  relationship between increasing oscillatory frequency of the head and the frequency of the CSF pulsations. To examine this frequency relationship further, the CSF locomotor pulsations were modeled as a sinusoidal wave, then a 95% confidence interval calculated for the model wave. The recorded CSF pressure consistently fit within the confidence interval, and there was significant overlap between the wave pattern of head oscillation and the modelled sinusoidal curves  sinusoidal changes Fig. A with meons Fig. B. Regresves Fig. C. Power ves Fig. D and a gves Fig. D.Figure The locomotor kinematics and CSF pressure had good congruence in the frequency domain Fig. B–D, but 22. While locomoting on the treadmill the alligators exhibited typical limb movements and undulations of the trunk, these resulted in the head sweeping through horizontal arcs . Sub-adult alligators, previously trained to walk on a treadmill, exhibited similar kinematic patterns before and after a fluid pressure sensor was implanted into their cranial subdural space. The CSF exhibited a clear and consistent pattern of pulsations linked to the cardiac cycle Fig. A, C, D; 18, support the conclusion that the CSF pressure pulsations reported in this study are generated by the movements of the alligators.There was a close congruence between the oscillatory movements of the alligator’s head and the pulsations in the CSF pressure Fig. B–D. Thes23. These suboccipital muscles would likely be contracting during head rotations, and previous experimental studies on alligators have shown that contraction of the myodural bridge alters the CSF dynamics16. Experimental manipulation of body cavity pressures in Alligator altered the CSF pressure24, it seems likely that changes in body cavity pressure, either ventilatory or independent, during locomotion would also influence the CSF. Additionally, the lateral undulations of the vertebral column that occur during terrestrial locomotion in crocodylians  which could alter CSF pressure.The physical bases for these movement-induced CSF pulsations are not clear. Crocodylians have a well-developed myodural bridge, a link between cervical skeletal muscles and the dura27 and rats29. The results of these four rodent studies have two key features in common: the onset of motion/locomotion was associated with a sharp increase in CSF pressure (to 3–4 × the resting level), and though the elevated CSF pressures recorded during motion/locomotion were quite variable, none of the published records contain anything like the sinusoidal curves reported from Alligator in the present study. Presumably, the absence of similar sinusoidal curves in the rodent studies reflect the substantial differences in body size and locomotor kinematics between rodents and Alligator. The sharp increase in CSF pressure reported at the onset of movement in rodents29 has been linked to a decoupling of cerebral arterial and venous volumes associated with increased cardiac output30. The CSF pressures recorded from locomoting alligators contain both sinusoidal waves, but also a gradual increase in CSF pressure  were obtained from the Louisiana Department of Wildlife and Fisheries. The animals were housed communally in a 29 m2 facility that featured three submerging ponds, natural light, and artificial lights on a 12:12 cycle. The facility was maintained at 30–33° C, warm water rain showers were provided every 20 min, which helped maintain the facility at > 75% relative humidity. The alligators were maintained on a diet of previously frozen adult rats. When the individual animals were removed from the enclosure, they were caught by noosing, then their jaws were taped using vinyl tape. The husbandry and use of the live alligators followed all applicable federal guidelines, and were approved by the IACUC of A.T. Still University , and are reported following the ARRIVE guidelines.Seven live sub-adult  American alligators (A treadmill (270 cm long × 61 deep × 46 wide) was fabricated out of steel beams, lined with stainless steel sheeting, and fit with a custom (538 cm long × 41 cm wide) tread . Each alligator was trained twice weekly to locomote along the length of the treadmill, initially with the treadmill off, subsequently with it moving in the range of experimental velocities (0.1–0.25 m/s). All training sessions were recorded using two digital video cameras , one providing a dorsal view and the other a head-on view. Once all of the alligators demonstrated stable locomotor patterns on the moving treadmill, the surgical experiments were initiated. One terminal surgical experiment was performed each week; the training schedule was maintained for all of the remaining animals.When the individual alligator was noosed for the surgical experiment is was induced to bite a bite pad, and the animal’s mouth was taped shut around the bite pad. Each individual alligator was placed on a stiff board (244 × 28 × 3.8 cm thick), which exceeded the maximum width and length of the alligators used for this study. Six 2.5 cm wide heavy duty straps (Northwest Tarp and Canvas) were used to secure the alligator to the board; the straps were tight enough to minimize movement of the animal but not tight enough to impede ventilation or circulation. With the alligator’s mouth held open by the bite pad, a laryngoscope was used to depress the gular valve and expose the glottis. A cuffed endotracheal tube was inserted into the larynx and connected to a custom anesthesia system that included a ventilator pump (Harvard Apparatus), Vaporstick anesthesia machine (Surgivet), isoflurane vaporizer (Surgivet), and Capnomac Ultima respiratory gas monitor (Datex-Engstrom). The alligators were maintained on a steady ventilatory pattern of 5 breaths per minute each with a tidal volume of 500 ml. Anesthesia was accomplished using 5% isoflurane. Two silver chloride surface cup electrodes , coated with a layer of conducting gel  were placed on the lateral surface of the animal, on either side of the heart. Meloxicam (at 0.2 mg/kg) was administered into the left triceps to serve as an analgesic.36. The lead coming off of the pressure transducer was sutured along the midline of the alligator to mid-body; neither the pressure catheter, transducer, nor the transducers lead, restricted the range of head or body movements of the alligator. The pressure transducer was coupled to a strain gauge amplifier , while the EKG electrodes were connected to a DC preamplifier  The outputs from these two amplifiers were sampled at 4 kHz, simultaneously with the carbon dioxide concentration from the respiratory gas monitor, using the MiDas data acquisition system (Xcitex Inc.).A surgical drill  was used to bore a 4.0 mm diameter hole through the dorsum of the alligator’s skull to expose the dura. A small incision was made in the dura to allow the passage of a pressure catheter. Surgical adhesive  was used to seal the dura around the catheter, then epoxy cement was added to fill the bored hole and secure the catheter to the skull. A fluid pressure transducer  was rested upon, and sutured to, the large osteoderm that covers the dorsum of the alligator’s neck. The pressure transducer, and the attached pressure catheter, were filled with a reptilian Ringers solutionThe experimental sequence for each alligator was similar. Once the alligator reached a surgical plane of anesthesia. The pressure catheter was implanted into the cranial sub-dural space and a series of baseline CSF, EKG, and (forced) ventilator cycles were recorded. With the animal still under anesthesia it was tilted 30° head-up and 30° head-down (separately), each over a 90 s duration divided into 30 s baseline, 30 s tilt, then 30 s recovery. The animal was allowed to recover from anesthesia, this is a slow (hours-long) process in alligators; the animal was actively ventilated with oxygen during recovery, but no drugs were administered to accelerate recovery. Once the animal was recovered enough to actively thrash against the restraining straps, the endotracheal tube and bite pad were removed (and the jaws again taped closed). The animal was then manually transferred to the adjacent treadmill and given time to adjust. Baseline recordings, now exclusively of CSF pressure, were taken while the animal was not moving on the treadmill. Once the alligator demonstrated coordinated voluntary movement, the treadmill was turned on and locomotor data collection commenced.Typically 3–4 locomotor sequences were recorded from each alligator (see below). Immediately after the last locomotor sequence the treadmill was turned off and the alligator allowed to remain (stationary) on the treadmill while post-locomotor CSF pressures were recorded. The animal was then returned to the surgical board, re-anesthetized, then euthanized through cardiac excision and exsanguination. Immediately after euthanizing the animal, the pressure catheter was removed and the catheter/transducer complex calibrated.The surgery and locomotor experiment were performed in the same small room which was maintained at > 85° F to prevent metabolic disruption to the alligators. An LED flash was located adjacent to the treadmill and provided one synchronization between the video and physiological data. A trigger switch controlled by one of the authors provided a second means of synchronizing the two data sets.The video records were analyzed using Kinovea (kinovea.org). The majority of the locomotor sequences recorded were not used for data analysis . Sequences were excluded if the alligator failed to perform at least 3 complete footfall (step) sequences, collided with the end or sides of the treadmill, was physically contacted by either researcher, or exhibited locomotor kinematics  that differed from those recorded during the final training sessions. Ultimately, 11 locomotor sequences were analyzed; these had a mean duration of 13.12 s (s.d. = 4.87). The physiological data were quantified with Midas. The data were exported into SpectraPlus (Pioneer Hill Software) for power spectral analysis, and were exported to EXCEL for analysis.To compare the frequency patterns of the head oscillation and CSF pressure traces, both traces were mathematically adjusted to have a mean of 0 and equal maximum amplitude. Using the timing data from the CSF recordings, a parameter optimization procedure was performed to find the best fit between a sinusoidal model [pulsation pressure = )))-amplitude offset] and the experimentally-recorded CSF pulsation pressure data. To compare the rates of change we used the unadjusted data records and compared the slopes of the head oscillation data (which yielded velocity of head rotation) to the slopes of the CSF pulsation curve (which yielded the rate of CSF pressure change).Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Video 1.


As torque increased, the CLBP group increased sEMG-torque coherence in more cranial ES regions, while the opposite was observed for the controls (p = 0.043). Individuals with CLBP show reductions in sEMG-torque relationships possibly due to the use of compensatory strategies and regional adjustments of ES-sEMG oscillatory activity.We quantified the relationship between spatial oscillations in surface electromyographic (sEMG) activity and trunk-extension torque in individuals with and without chronic low back pain (CLBP), during two submaximal isometric lumbar extension tasks at 20% and 50% of their maximal voluntary torque. High-density sEMG (HDsEMG) signals were recorded from the lumbar erector spinae (ES) with a 64-electrode grid, and torque signals were recorded with an isokinetic dynamometer. Coherence and cross-correlation analyses were applied between the filtered interference HDsEMG and torque signals for each submaximal contraction. Principal component analysis was used to reduce dimensionality of HDsEMG data and improve the HDsEMG-based torque estimation. sEMG-torque coherence was quantified in the δ(0–5 Hz) frequency bandwidth. Regional differences in sEMG-torque coherence were also evaluated by creating topographical coherence maps. sEMG-torque coherence in the δ band and sEMG-torque cross-correlation increased with the increase in torque in the controls but not in the CLBP group ( The neural origin of torque fluctuations has been a topic of interest for many years1. Previous studies have proposed that the variability in the times that motoneurons discharge action potentials could be a potential determinant for the accurate control of muscle force2. This variability could be determined by independent and common synaptic inputs received by motor units and/or by the nonlinear input–output properties of individual motoneurons1. However, recent studies have shown that the neural drive to the muscle  in low frequencies (< 10 Hz) is mainly relevant for torque generation as it reflects the common synaptic input received by the motor unit pool5. This happens because a group of activated motoneurons can act as a very selective filter, which eliminates all the independent components, partly linearizes the input–output motoneuron behavior and amplifies the common component4. Thus, the control of muscle force depends mainly on the amplitude of the low-frequency oscillations that reflect shared (common) synaptic input1.When executing a steady submaximal isometric contraction, the applied force or torque is not perfectly accurate but rather, small oscillations occur around the target torque value6. These central and peripheral signals are sent to the somatosensory cortex via spinal, subcortical and cortical neural pathways, where they are integrated along with other somatosensory, visual, and vestibular information, resulting in the perception of applied forces7. A descending motor command is then sent to the tissues involved, resulting in an appropriate action8. The proprioceptive system ensures smooth force production through this interactive process, that requires constant monitoring and modulation of ascending and descending information based on the proprioceptive input, motor output, perception, and the resulting action8. Therefore, any perturbation to these systems, can affect the control of muscle force.Smooth force production depends highly on our proprioceptive senses of effort and force, with the first being generated centrally (via corollary discharges of the descending motor command), and the latter derived from peripheral sensory receptors 9. For example, studies have shown that people with subacromial impingement syndrome10, knee osteoarthritis11, neck pain12 and chronic low back pain (CLBP)15 display reduced force steadiness. Impaired lumbar sensorimotor control, could possibly lead to suboptimal tissue loading  which can induce further pain/injury and/or perpetuation of symptoms9. However, the neuromuscular mechanisms responsible for poorer muscle force control when people have pain have not been fully established.Pain can affect the ability to control muscle force, possibly due to altered afferent input from the painful area and/or neuroplastic changes in the central nervous system 16. Taking this a step further, Moon et al., (2014) showed that the low frequency component of the rectified interference sEMG is also coherent with the oscillations in force at low frequencies which contributes to force variability17. Nevertheless, conventional sEMG recordings present numerous limitations since factors such as cross-talk, amplitude cancellation, electrode position and volume conductor effects (among others) can affect the relationship between sEMG and torque19. Hence, it is not surprising that only weak correlations have been observed between force and sEMG5.Many studies have attempted to assess the neural origin of fluctuations in muscle force by assessing surface sEMG and torque/force relationships. For instance, Yoshitake and Shinohara, (2013) previously demonstrated that the temporal characteristics of force fluctuations at low frequencies (< 5 Hz) are not only correlated with the motor unit discharge times (common drive), but also with the low frequency component of the rectified interference sEMG5. Nevertheless, the applicability of HDsEMG decomposition is limited to certain experimental conditions and can be only achieved for certain muscles20. For example, the identification of motor units from trunk muscles  is not currently possible with HDsEMG. Although intramuscular recordings could be potentially used to assess motor unit firing patterns from trunk muscles, their selectivity does not allow identification of a sample of motor units that is large enough to obtain an accurate representation of the exerted torque. One alternative to overcome these issues is taking advantage of HDsEMG's high spatial sampling resolution. Staudenmann et al.21 previously showed that HDsEMG can improve sEMG-based force estimations by 25%. This improved estimation is enabled by employing dimensionality reduction techniques such as Principal Component Analysis (PCA) which reflects the common variability between the multiple sEMG signals recorded with HDsEMG, providing a unique signal which can explain most of the variance in exerted torque22. In addition to PCA, HDsEMG has the possibility, not yet exploited, of generating a topographical map assessing relationships between torque and sEMG from the regions covered by the electrode grid. This would allow to determine whether certain muscle regions contribute more to the exerted torque than others. In the case of CLBP, several studies have revealed variations in lumbar ES amplitude distribution27, and in particular, studies have shown that people with CLBP often display less activity in the more caudal regions of the lumbar ES during static-fatiguing and dynamic lumbar extension tasks27. It is possible that regional variations in sEMG-torque relationships are responsible for poorer control of force during visuomotor torque-matching tasks, yet this has never been examined.Recent studies have shown that high-density electromyography (HDsEMG) can improve our understanding of the mechanisms responsible for the control of muscle force with HDsEMG motor unit decomposition. Indeed, HDsEMG recordings allow the identification of a large number of motor units and their discharge characteristics enable an accurate prediction of the resultant torqueThe aims of this study are to (1) quantify the relationship between HDsEMG oscillations and isometric trunk-extension torque oscillations in both time  and frequency domains  and (2) examine and compare regional differences in sEMG-torque coherence of the ES muscle in individuals with CLBP and asymptomatic controls. It was hypothesized that individuals with CLBP will show a weaker correlation between HDsEMG and torque and a more focalized distribution of HDsEMG-torque coherence of the lumbar ES muscle.28. All experimental procedures were conducted according to the Declaration of Helsinki. Data collection was performed between April 2019 and January 2020 at a laboratory within the Centre of Precision Rehabilitation for Spinal Pain (CPR Spine), University of Birmingham, UK. All participants attended one laboratory session and their written informed consent was obtained prior to their participation. All participants were encouraged to address any concerns or questions related to the study before signing the informed consent.This case–control, cross-sectional study was approved by the Ethics Committee of the University of Birmingham  and work is reported in accordance with the STROBE guidelines29, from mean (SD) variable error values of individuals with CLBP and asymptomatic controls during a multidirectional isometric tracking task at 90° and at 20–40% of maximal voluntary contraction (MVC)], and a potential 5% loss of data due to signal quality or participant withdrawal.Fifteen individuals with CLBP  and 15 asymptomatic controls  from the University of Birmingham student and staff community were recruited for this study, by means of information leaflets posted around the University of Birmingham and posts on social media. The sample size was calculated based on an α of 0.05, a power of 0.8, a moderate effect size of (f) 0.28 , the motor performance characteristics (variations in CoV and SD of torque) and the self-reported outcome measures . The level of statistical significance was set to p = 0.05.All data analyses were performed on SPSS Statistics, version 27 . The Shapiro–Wilk test was used to test the normality of the data and the assumption of homogeneity of variance was assessed with a Levene test. These assumptions were met, and thus parametric tests were applied. Descriptive statistics were used to analyze the data and the results are expressed as means and SDs. Anthropometrics, NPRs scores, questionnaire scores and isometric back extension MVC between groups were compared with independent 55. As anticipated, the controls did not report low back pain. None of the participants reported an increase in their level of pain intensity after performing the contractions.All 30 participants  successfully completed the study, and their characteristics are reported in Table p = 0.350, ηp2 = 0.031). Overall, individuals with CLBP had significantly higher CoV and SD of torque than the asymptomatic controls during the two torque levels . The average MSE values for both groups and torque levels were as follows: Controls at 20%MVC and 50%MVC: 0.18(0.22) % and 1.44(1.15) % respectively, CLBP at 20% MVC and 50%MVC: 0.69(0.79) % and 2.43(2.06) % respectively.Both groups exerted similar levels of isometric peak torque during the MVC of isometric back extension Table . The meap = 0.060, ηp2 = 0.121). The mean values for both groups and torque levels were as follows, controls at 20%, 50%MVC: 37.8(19.9)% and 60.5(17.9)% respectively, CLBP at 20%, 50%MVC: 52.1(15.9)%, 76.1(29.9)% respectively. Both groups presented similar levels of RA-ES co-activation during the submaximal contractions . The average RA-ES co-activation values for both groups and torque levels were as follows, controls at 20%, 50%MVC: 130.4(128.3)% and 84.9(62.5)% respectively, CLBP at 20%, 50%MVC: 114.9(89.9)%, 100.5(70.2)% respectively.The normalized spatial mean of the RMS values for the lumbar ES did not differ between groups during the two torque levels .No significant correlation (Pearson’s r) was observed between any of the sEMG, motor output (torque production) and self-reported (pain and function) parameters (In this study we aimed to (1) measure the relationship between HDsEMG oscillations and isometric trunk-extension torque in both time and frequency domains, using cross-correlation and coherence analysis respectively, and (2) identify and compare regional differences in sEMG-torque coherence of the lumbar ES in individuals with CLBP and asymptomatic controls. sEMG-torque coherence in the δ band and sEMG-torque cross-correlation increased with the increase in torque for the asymptomatic controls, but this was not observed for the CLBP group. Additionally, the CLBP group had higher sEMG-torque coherence in more cranial regions of the lumbar ES at 50%MVC, while the opposite was observed for the asymptomatic controls . Interestingly, the level of lumbar ES activity (HDsEMG amplitude), and the co-activation levels did not differ between groups during the tasks.56.We quantified the relative power only in the δ (0–5 Hz) frequency bandwidth from the interference sEMG and torque signal since torque fluctuations during isometric contractions are mainly explained by the effective neural drive to the muscle, i.e., low-frequency component (< 10 Hz) of the neural drive to the muscle47, as seen in the asymptomatic group, the CLBP group failed to increase sEMG-torque coherence with the increase in torque. This finding is also supported by the cross-correlation results, showing that in the CLBP group, the net increase in torque was not accompanied by an increase in the contribution of the lumbar ES muscle to the resultant torque fluctuations. Given that the low-frequency component of the rectified sEMG is associated with the variability in motor unit firing rate and hence torque oscillations57, correlation and coherence between sEMG and torque, should have been higher in the CLBP as they had higher variability in torque fluctuations compared to the asymptomatic controls. However, this was not observed, as at both torque levels, cross-correlation and coherence was similar for the CLBP group. Additionally, given that both groups exerted a similar amount of torque during the task, the individuals with CLBP could have compensated by activating synergistic muscles. Under the influence of pain, the central nervous system can find a new motor strategy to achieve a specific task  and due to the redundancy of the trunk motor control system, people with CLBP often display substantial reorganization of their motor control strategies58. This can allow people with CLBP to adopt different muscle recruitment solutions to achieve a specific outcome and possibly avoid pain. Importantly, these changes can be explained by alterations of the descending drive to the muscles, but also by changes in motor planning 59. In the present study, individuals with CLBP had higher levels of Kinesiophobia (higher scores in TSK) compared to the asymptomatic controls. Thus, it is possible that the CLBP group experienced more fear as the torque increased, leading to the use of compensatory strategies, which likely involved the hip extensors, and this would explain why we did not observe an increase in the sEMG-torque coherence and cross-correlation values in the δ band . This is also further supported by a previous observation, showing that initially pain-free individuals with high pain-related fear adopt avoidant strategies during common reaching movements shortly after the induction of delayed-onset muscle soreness60. However, it should be noted that this is speculative, more so considering that the average TSK score of 34.7 for the CLBP group is still considered low. Future studies could investigate this further. Interestingly, the levels of lumbar ES activation and ES-RA co-activation were similar between the two groups. This shows that RMS amplitude alone  could not explain any differences between the two groups in the execution of the torque steadiness task, but the proposed coherence analysis gave us better insight into the actual contribution of the trunk extensor muscles to the resultant torque.Interestingly, even though an increase in torque is commonly accompanied with an increase in δ band coherence61 and that altered distribution of input to the motoneurons can change motor unit recruitment patterns during pain62, we investigated whether regional differences in sEMG-torque coherence also existed between people with CLBP and asymptomatic controls. Differences in topographical representation of coherence were observed at 50%MVC, since the lumbar ES coherence centroid in the CLBP group was displaced more cranially (suggesting higher coherence in the upper region of the muscle), while in the asymptomatic controls, the centroid of coherence was displaced towards more middle-caudal regions of the lumbar ES. This finding likely suggests that that torque is less represented in the caudal region of the muscle in individuals with CLBP at 50%MVC, since the correlation between sEMG and torque is the lowest in this region. Since common synaptic input is reflected by δ band coherence, this could suggest that individuals with CLBP show reductions in sEMG-torque relationships due to a more focalized distribution of common synaptic input (HDsEMG-torque coherence) of the lumbar ES and possibly a higher influence of independent synaptic inputs on the lumbar ES motor unit pool, at least when they exert higher forces. However, these findings should be confirmed with sEMG-torque coherence, based on sEMG decomposition analysis, as the interference sEMG signal is influenced by several factors19. From a clinical perspective, it should be also noted that this more focalized contribution of muscle fibers to the resultant torque, imply that those specific muscles fibers  will be the main contributors to the resultant torque during extension. This could possibly lead to an uneven distribution of load, thus, overloading of those specific muscle fibers61. Such motor behaviour could possibly play an important role in the perpetuation of CLBP symptoms.Moreover, given that regional differences in myoelectric activity of lumbar ES is a common finding in people with CLBP15 and further support the notion that the control of muscle force (measured either as force accuracy and/or force steadiness/variability) is impaired in individuals with CLBP. Trunk muscle function impairments are common in people with CLBP, and these can be more pronounced when motor control is challenged by tasks that require high precision, such as the one used in the current study15. These impairments, including reduced torque steadiness, are likely attributed to the numerous functional and structural neuroplastic changes that can occur at multiple levels of the nervous system  when people have pain9. For example, proprioceptive acuity is reduced in people with CLBP9, and this can be related to suppression of proprioceptive input by nociceptive afference and/or altered afferent input due to changes in the mechanical properties of passive structures 8. Additionally, it could be related to structural changes, such as the reduction of white matter integrity of the superior cerebellar peduncle, which is an important brain area for the transmission of proprioceptive input to higher centers63. Importantly, previous studies have also shown that regions of the motor cortex that are related with the control of specific trunk muscles are also reorganized in people with CLBP , and this is likely related to a loss of discrete control of paraspinal muscle fascicles65. Collectively, these neuroplastic changes observed in people with CLBP could explain their impaired capacity to control trunk muscle force accurately. Compensations for proprioceptive deficits and/or other neuroplastic changes can be reflected as alterations in the efferent motor response . Therefore, it is relevant to unravel the motor control strategy that the CLBP group utilized to exert the requested amount of torque during the torque steadiness and MVC tasks.As anticipated, people with CLBP had reduced trunk extensor muscle torque steadiness, compared to asymptomatic controls Fig. . These f66. Previous research has shown that pain decreases the ability to exert maximal forces68, due to nociceptive-induced inhibition to agonist muscles69. However, these findings are mainly observed in upper or lower-limb muscles, which produce torque around a single joint 67 or across two joints 68. On the contrary, trunk extension is a multi-joint movement that involves concurrent movement of the lumbar spine, pelvis and hips70. Therefore, people with CLBP could have performed the MVC by compensating with the large hip extensor muscles , which can influence both the hip and pelvis, thereby minimizing the activation of their lumbar extensors. Although, the thighs and pelvis were stabilised with straps, it is not possible to completely prevent such compensatory strategies. These factors could have allowed the participants to perform the MVC with a higher contribution of their hip extensors, compared to their lumbar extensors (possibly due to pain and/or fear). As previously shown, when the thigh is stabilised, the hamstrings and the gluteus maximus rotate the pelvis posteriorly which likely “disengages” and counteracts lumbar extension71. Thus, during the MVC, people with CLBP could have exerted torque by extending their hips with their pelvis tilted posteriorly . It could be also argued that people with CLBP managed to exert a similar amount of torque during the MVC by greater activation of deeper lumbar extensor muscles, such as the multifidus muscle, however, this is unlikely as this muscle’s main function is to stabilize the lumbar spine and is more commonly inhibited in people with CLBP72.Trunk extensor isometric muscle strength did not differ between groups, which is consistent with the results of some previous studies75 and/or sEMG parameters76 in people with musculoskeletal pain. Some possible reasons for the lack of correlation between these parameters are the following: (1) the fact that people with CLBP present a wide variety of neuromuscular responses and that there is substantial variability even considering one type of neuromuscular adaptation, (2) the characteristics of the individuals with CLBP recruited for this study , and (3) the variability in the questionnaire data due to their subjective nature. It is likely that each participant with CLBP used a different strategy to maintain a steady force during the task regardless of the level of pain and/or changes in function77. However, this should be confirmed in future studies which incorporate recordings from multiple muscles during similar tasks in order to obtain greater insight of the potential compensatory strategies utilized by people with CLBP.Lastly, it should be noted that there was no significant correlation between the sEMG parameters, the motor output (torque production) parameters and the self-reported measures of pain and function. This makes it difficult to explain the observed changes in the sEMG parameters and means that the neuromuscular mechanisms responsible for the poorer trunk muscle torque steadiness observed in individuals with CLBP, warrant further investigation. However, this is not a surprising finding, as many studies do not report any significant correlation between motor output and self-reported measures of pain/function24. It is likely that a sample of people with more severe CLBP would present with even higher deficits, or other neuromuscular adaptations which could negatively impact on the ability to control torque output in a steady way. Additionally, this study did not measure possible compensatory patterns by using kinematic markers and/or recording sEMG activity of synergist muscles. Another limitation that should be considered is that the rectified interreference sEMG is only a rough estimator of the neural drive to the muscles, as it is influenced by several factors, including amplitude cancellation 78. Based on the findings of a simulation study, amplitude cancellation can distort the spectrum of the rectified sEMG signal and thus have an influence on the results78. This problem is commonly solved by basing the analysis on the identification of motor unit spike trains , rather than on sEMG. However, HDsEMG decomposition is currently challenging on signals collected from the lumbar ES likely due to factors such as the (1) complexity of lumbar anatomy 79, and (2) the volume conductor properties of the lower lumbar area 20. These factors can reduce the discriminative information in the action potential waveforms of different motor units, and thus limit the applicability of decomposition algorithms20. Therefore, we based our analysis on the rectified sEMG signal. Nevertheless, the correlation between the sEMG signal  and torque was ≈ 0.4 for both groups and torque levels, likely suggesting a moderate correlation between sEMG and torque oscillations. Studies employing motor unit recordings usually report higher discharge rate vs torque cross-correlation values (approximately 0.75–0.80)5. However, this is usually seen in smaller, single-joint muscles . It is likely that these correlations would be reduced in larger muscles like ES, however, this is yet to be determined. Lastly, another limitation that needs to be addressed is the calculation of co-activation based on non-normalised RA and ES RMS values, which might have provided less reliable co-activation estimates due to inter-subject anatomical differences . Nevertheless, in our previous study, co-activation was calculated based on normalised RA and ES RMS values during a dynamic isokinetic fatiguing task and again we did not observe differences in co-activation between people with CLBP and asymptomatic controls23. On top of this, whether absolute or normalised sEMG-RMS amplitudes are favorable in the interpretation of co-activation index remains unclear80. Thus, we believe that similar findings would have been observed, even if the RMS values were normalised during the calculation of co-activation ratios.A limitation of this study is that the sample consisted mainly of young, active individuals with low pain intensity and mild disability. This likely limits the generalisability of the results. However, even with relatively low levels of disability and pain intensity we were still able to identify impairments in this group, although it should be noted that there was variability between individuals with CLBP, as has been commonly observed previouslyThis study uniquely demonstrates that individuals with CLBP fail to increase the common fluctuations in torque and HDsEMG activity, when exerting higher lumbar extension forces. This reduced ability to alter the sEMG-torque relationship, is likely due to regional adjustments of ES-sEMG oscillatory activity  and the use of compensatory trunk extension patterns during this task. The current study contributes to the existing body of knowledge and further supports the notion that individuals with CLBP have poorer control of their trunk extensor muscles compared to asymptomatic individuals.We would like to acknowledge the contribution of Bikinis Nikolaos, Petrakis Stylianos, Gkioka Afroditi and Tsimpolis Dimitrios, students at the School of Sport, Exercise and Rehabilitation Sciences of the University of Birmingham, for their help with the experimental procedure. We would also like to thank the volunteers participating in this study.


We compared methods used with current recommendations for synthesizing harms in systematic reviews and meta-analyses (SRMAs) of gabapentin.We followed recommended systematic review practices. We selected reliable SRMAs of gabapentin  that assessed at least one harm. We extracted and compared methods in four areas: pre-specification, searching, analysis, and reporting. Whereas our focus in this paper is on the methods used, Part 2 examines the results for harms across reviews.We screened 4320 records and identified 157 SRMAs of gabapentin, 70 of which were reliable. Most reliable reviews  reported following a general guideline for SRMA conduct or reporting, but none reported following recommendations specifically for synthesizing harms. Across all domains assessed, review methods were designed to address questions of benefit and rarely included the additional methods that are recommended for evaluating harms.Approaches to assessing harms in SRMAs we examined are tokenistic and unlikely to produce valid summaries of harms to guide decisions. A paradigm shift is needed. At a minimal, reviewers should describe any limitations to their assessment of harms and provide clearer descriptions of methods for synthesizing harms. GuidelinMeta-research has shown that primary studies and systematic reviews use poor methods to assess harms and report them poorly.–21,5–12 Our objectives in this study were to assess whether reviews: used methods described in these guidelines, used appropriate sources of evidence, and applied appropriate methods to synthesize harms. We compared results for harms in a second paper.We selected gabapentin as a case example because it was likely that there would be multiple systematic reviews to compare Additionally, gabapentin is used widely for a range of conditions, of which most prescriptions are off-label.2.We prespecified eligibility criteria, the search strategy, items for extraction, and the comparison of methods with recommendations. As part of a PhD dissertation proposal, the protocol was reviewed by a committee and is attached as 2.1.To be included in our study, we required that reviews: (i) be systematic reviews or meta-analyses; (ii) examine gabapentin for one of its commonly prescribed conditions(on- or off-label), including: alcohol dependence, epilepsy, pain , psychiatric disorders , restless leg syndrome, and vasomotor symptoms, and; (iii) have any results for harms ; and (iv) be reliable in methods . We defined systematic reviews as articles that either (i) self-identified as a systematic review or metanalysis, or (ii) followed a structured methodology to synthesize research, as per the Institute of Medicine’s definition of a systematic review.We focused on reliable reviews, which would be the “best case scenario” for agreement in methods for assessing and reporting harms. We excluded reviews published only as abstracts or for which a full text was unavailable because it is not possible to assess the reliability or methods of a review based solely on an abstract.2.2.We searched PubMed, EMBASE, Epistemonikos, and the Cochrane Database of Systematic Reviews from 1990 to September 17, 2020 without any language restrictions (gabapentin was first approved for use by the United States Food and Drug Administration in 1993). We developed a search strategy with informationists from Johns Hopkins Welch Medical library .We used EndNote then Covidence for de-duplication and screening. Two authors (RQ and TR) screened titles and abstracts, and full texts, independently and resolved disagreements through discussion. We piloted the screening with 100 and 50 records at the title/abstract and full-text levels, respectively.2.3.We extracted all details about the methods used in systematic reviews to assess harms, grouped as follows: approach/planning, searching, analysis, and reporting. Specific items extracted for each of these domains can be found in Both reliability assessment and data extraction were done by two reviewers using single extraction with verification, which isas accurate as independent data extraction but less resource intensive. We used 2.4.post hoc subgroup analyses based on (i) whether reviews pre-specified any harms and (ii) whether the primary purpose of the review was to assess questions related to harms or benefits. Reviews with pre-specified harms might have included a search for studies of those harms. By contrast, reviews focused on potential benefits might not include a search for harms at all. Thus, we explored differences in the searches and types of evidence used to address these different types of research questions.We qualitatively describe and compare the methods used for harms across all studies that met our inclusion criteria. We performed two We used Microsoft Excel and Stata 15 to tabulate all results.3.3.1.3.2.3.3.No reviews stated they followed any guidance for synthesizing harms .Most reviews  aimed to assess the evidence for both potential benefits and harms—often with a greater focus on benefit—while 10/70 (14%) reviews focused solely on assessing harms. A hybrid approach—wherein at least one pre-specified harm was addressed alongside additional harms identified during the review—was taken by 18 of 70 (26%) reviews. Nearly equal numbers of reviews either did not pre-specify any harms of interest  or assessed only pre-specified harms  . Among tReviews often assessed multiple types of harms, most commonly specific ‘unique harms’ such as dizziness, somnolence, and weight gain . Reviews also commonly assessed harms using proxies such as “drop-out” or “loss-to-follow-up due to harms”  .3.4.Only 22/70 (31%) reviews performed supplemental searches for unpublished studies or data from regulatory agencies or industry. We found that not many reviews searched for types of studies used only to assess harms  . Our sub3.5.Of the 70 included reviews, 26 (37%) assessed harms only descriptively , 19 (27%) assessed all harms quantitatively, and 25 (36%) assessed harms using both descriptive and quantitative methods . Where qOf the 70 reviews, 44 (63%) performed a quantitative analysis of at least one harm. Eleven (25%) of the 44 conducted a network meta-analysis for harm or a proxy for harm . Only thMost reviews specified whether they used fixed-effect or random-effects models used for analysis. Only three (7%) reviews did not report what type of meta-analysis they conducted for harms: 32/44 (73%) reported at least one random-effects analysis and 15/44 (34%) reported at least one fixed-effects analysis for harms. Although the defaults for meta-analysis in most statistical programs used to conduct meta-analysis  are inverse-variance models, which are often biased for analyzing rare events,–39 we fo3.6.Harms reporting was often unclear and inconsistent. Most reviews  included a statement about harms in the abstract; either about specific harms  or a more general statement about the potential for harm  . Many reMost reviews  included a statement about limitations specifically for harms. These limitations were commonly about adverse effects being poorly or inconsistently reported among included studies, trial study designs  being insufficient for identifying harms, limiting included studies to trials, and not searching uncontrolled or unpublished literature.4.The desire to address harms in systematic reviews has led to tokenism. Guidance indicates that all reviews should assess harms,,7,22 butTo address these challenges to synthesizing information about harms, a paradigm shift is needed. Cochrane and other producers of systematic reviews should reconsider how best to guide review authors to assess harms in systematic reviews that are designed primarily to assess potential benefits. We believe that systematic reviews specifically focused on harms are needed. Because many drugs are used for multiple indications, systematic reviews of harms that are limited to specific indications will lead to incomplete evidence. When appropriate, Consciously separating reviews of benefits and reviews of harms could increase validity, avoid unintended overlap, and reduce overall burden on systematic reviewers.Appropriate methods to review harms differ from appropriate methods to assess potential benefits. For example, multiple study types should be considered when answering questions about harms, so systematic reviewers should not exclude observational data at the outset of the review process.,7,22 WhiSecond, the reporting of analyses of harms requires greater detail in reviews. Reviewers should specify how the analyses are conducted (whether they were assessed the same or differently from benefits) and key assumptions such as handling of rare events and missing data.,40 CommoThird, and perhaps most importantly, reviewers should discuss the choice of selection criteria for assessing and reporting harms . SelectiReviews of harms require resources and information that are unavailable to many academic researchers. In this regard, responsibility for conducting and publishing systematic reviews of harms may fall on government organizations. For pharmaceuticals and devices, regulators such as the US Food and Drug Administration are well-positioned to conduct these reviews properly because they have access to individual participant data from trials, and they have epidemiologists and biostatisticians skilled at analyzing real world data on harms. Although regulators might be unable to undertake all the research they would like to conduct, publishing studies they do conduct would be valuable contribution to policy and practice.Lastly, reviews should describe the limitations of any harms assessments. Following every recommendation above might be impossible or unnecessary in every review. so if recommended methods are not followed, then reviewers should provide a rationale and report any associated limitations so that readers can evaluate their confidence in the results. Peer reviewers and journal editors should strive to ensure that reporting guidelines are followed and that systematic reviews do not present overly confident summaries of harms when they use unreliable methods.5.Consistent with the results of previous research, the current methods of assessing harms in the systematic reviews we examined does not produce valid evidence about harms. The underlying issues we identified demand a paradigm shift rather than incremental change or further guidance. Harms are currently included as tokenistic outcomes in reviews for the sake of assessing the “balance” of benefits and harms. We propose that reviewers fundamentally revise how we approach harms from review conception through resource collection and analysis to reporting.4321


In recent years, under the pressure of resource shortage and white pollution, the development and utilization of biodegradable wood-plastic composites (WPC) has become one of the hot spots for scholars’ research. Here, corn straw fiber (CSF) was chosen to reinforce a poly(lactic acid) (PLA) matrix with a mass ratio of 3:7, and the CSF/PLA composites were obtained by melt mixing. The results showed that the mechanical properties of the corn straw fiber core (CSFC) and corn straw fiber skin (CSFS) loaded PLA composites were stronger than those of the CSFS/PLA composites when the particle size of CSF was low. The tensile strength and bending strength of CSFS/CSFC/PLA are 54.08 MPa and 87.24 MPa, respectively, and the elongation at break is 4.60%. After soaking for 8 hours, the water absorption of CSF/PLA composite reached saturation. When the particle size of CSF is above 80 mesh, the saturated water absorption of the material is kept below 7%, and CSF/PLA composite has good hydrophobicity, which is mainly related to the interfacial compatibility between PLA and CSF. By observing the microstructure of the cross section of the CSF/PLA composite, the research found that the smaller the particle size of CSF, the smoother the cross section of the composite and the more unified the dispersion of CSF in PLA. Therefore, exploring the composites formed by different components of CSF and PLA can not only expand the application range of PLA, but also enhance the application value of CSF in the field of composites. Petroleum-based plastics have flourished due to their light weight, low cost, simple process, dramatic mechanical properties and versatility, which become the raw materials for most of the current material products ,2,3. HowPoly(lactic acid) (PLA) has been proven to be an ideal substitute for petroleum-derived polymers, and has become one of the representatives of petroleum-derived polymers, resulting from its complete biodegradability, perfect bio-compatibility, and processability. In addition, raw materials for the preparation of PLA come from renewable crops, such as corn, sugarcane, and cassava. However, the application of PLA is limited by its high cost, inherent brittleness and poor thermal stability, among other disadvantages. ,14. TherStraw is one of the most abundant natural renewable materials in the world. Fibers sourced from straw have the advantages of being renewable, biodegradable and low cost. In recent years, straw fiber has received much attention as a reinforcing agent for composite materials, mainly divided into crops such as corn straw, wheat straw, rice straw and barley straw ,16,17. AUnlike other biomass fibers, CSF can be divided into the following two parts : corn stThe performance of fiber-reinforced composites in general depends on various factors, e.g., fiber size, interfacial compatibility between fiber reinforcement and the matrix, the aspect ratio of fibers and components of corn straw fiber. Due to the large polarity difference between natural fibers and PLA, the interfacial bonding between the two phases is poor and has a significant effect on the performance of composite materials, so CSF treatment is required ,31. HuanConsidering the physical characteristics and technological conditions of CSF and PLA, this paper uses twin-screws to melt blend CSF and PLA with different components and particle sizes to prepare CSF/PLA composites. The mechanical properties, crystallization and thermal properties of CSF/PLA composites prepared with different components were compared by means of infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and thermogravimetric analysis (TG).The testing of tensile and bending properties is of great significance to the application fields and application values of composite materials. The mechanical properties analysis of the CSF/PLA composites (weight mass ratio 3:7) with different particle sizes and compositions are shown in −1, attributed to the -OH group. The absorption peak at 2822–2940 cm−1 was the typical stretching vibration peak of -CH (methyl and methylene groups) on cellulose and hemicellulose in CSF, and the absorption peak at 1742 cm−1 was attributed to the stretching vibration peaks of the ester carbonyl C=O in the CSF hemicellulose and the -CH deformation peaks (symmetric and asymmetric bending peaks) at 1385 and 1368 cm−1. The absorption bands at 1080 and 863 cm−1 were assigned to the stretching vibration of C–O–C and C–C, respectively [−1 and 1752 cm−1 can be observed. The absorption peaks of -CH3 at 1353 cm−1 and 1071 cm−1 are attributed to the C–O–C stretching vibration. The absorption peak at 3350 cm−1 was significantly weakened after the straw fiber was blended with PLA, indicating the formation of a hydrogen bond between -OH of straw fiber and C=O of PLA [−1 of the straw fiber/PLA composite weakens significantly compared with that of the PLA, indicating that chain scission and degradation occurred during the preparation of the composites [−1 decreased, indicating that the molecular chain scission and degradation occurred at the ester group of the PLA.In order to analyze the chemical functional groups of the different PLA composites, the FT-IR curves of PLA before and after mixing with different components of CSF are shown in ectively . From thmposites . The carThe contact angle of CSF/PLA composites were determined, as shown in The water absorption rate of the CSF/PLA composites with different components and particle sizes changes, as shown in The TG and DTG curves of the CSF/PLA composites are shown in Tcc) of the CSF/PLA composites shifts to the low temperature region, which indicates that the crystallization ability of the material is improved. As shown in Crystallization of the diverse CSF/PLA composites were examined by a differential scanning calorimeter (DSC). The secondary heating curve is shown in The SEM images of the tensile fracture surface of the composites are shown in In studying the feasibility of corn stalk fibers as the reinforcement of CSF/PLA composites, the effect of fraction and fiber size on the mechanical properties of the resultant composites was investigated. The particle size of straw directly affects the tensile and bending properties of the composites. The 80–120 mesh SCFS/PLA composites’ tensile strength is 49.51 MPa and bending strength is 85.39 MPa. As observed by scanning electron microscopy, the fracture morphology was smoother due to the uniform distribution of the fibers. Due to the pore structure of CSFC, the composites containing CSFC components can greatly enhance the compatibility between PLA and CSF. The contact angle test and the water absorption test show that the CSF/PLA composites with a particle size of more than 80 mesh have better hydrophobicity. This result offers a new solution for the renewable utilization of corn straw fibers and improves their economic benefits and utilization value. At the same time, this paper could provide new insights into the use of corn straw for PLA matrix reinforcement.PLA, grade 4032D, NatureWorks Co., Ltd. ; corn straw fiber, self-made; 20–120 mesh CSF.Corn straw fiber was pulverized with the multi-functional pulverizer . The CSF were separated by sieves with different mesh numbers. The CSF with particle sizes larger than 40 mesh was air-selected by the blower to separate CSFS and CSFC. Due to the differences in density, CSFC was blown away and collected under the action of the blower, while CSFS was settled due to gravity. Then, the CSFS with different meshes was pulverized with the multi-functional pulverizer. CSF, CSFS and CSFC with different particle sizes were screened by sieve and grouped as shown in the PLA and screened CSF of different groups were mixed evenly according to the mass ratio of 3:7. The uniformly mixed raw materials were placed into the twin-screw extruder  to obtain CSF/PLA composites . By the micro injection molding machine , the dumbbell-shaped composite sample was obtained by injection molding. The injection temperature was 190 °C, the injection molding pressure was 0.8 MPa, and the heating time was 400 s. The appearances of different PLA composites are shown in The CSF/PLA composites’ physical and mechanical properties were measured by a testing machine . According to the national standards (GB1040.3-2006), the tensile speed is 6 mm/min and the span is 40 mm. The bending test was performed at three-point bending head down speed of 6 mm/min and span 45 mm .−1.The thermogravimetric analysis  was performed on 2~5 mg of the CSF/PLA composites under an argon atmosphere. The sample was conducted from 30 °C to 700 °C, with a heating rate of 10 °C·min−1, held for 5 min to eliminate thermal history, and then cooled at a rate of 10 °C·min−1 to −20 °C. The sample then was reheated to 210 °C at 10 °C·min−1 as the second cycle to identify the impact of CSF on crystallization and thermal behavior of composites. The crystallinity of CSF/PLA composites is calculated by Formula (1), which is as follows:ΔHcc is the enthalpy of cold crystallization; ΔHm is thev; ΔHm0 is the melting enthalpy when the PLA is completely crystallized, which is 93.7 J·g−1; ω is the percentage of polylactic acid in the composite material [A differential scanning calorimeter  was used to examine the thermal properties of the CSF/PLA composites. The sample was conducted in the range of 25~200 °C at a heating rate of 10 °C·minmaterial .A scanning electron microscope  was used to observe the morphology of the fracture section of the CSF/PLA composites, the accelerating voltage was 10 kV. The samples were soaked in liquid nitrogen for more than 15 min and then after being fractured, the fractured surfaces were sputter-coated with gold before imagining.−1.The chemical functional groups of CSF and PLA before and after compounding with each other were examined by Fourier transform infrared spectroscopy . The FTIR spectra of the samples were conducted between the range of 500 and 4000 cmW is the water absorption percentage (%); m1 (g) and m2 represent the quality of the sample before and after immersion, respectively [A water absorption test was performed on CSF/PLA composites with 20 × 10 × 4 mm dimensions. Each sample was dried at 30 °C for 72 h. The weight of the samples before and after immersion in distilled water for 24 h were recorded at 25 °C. Five samples for the same component were weighed to calculate its average value. The formula for calculating the water absorption rate of the sample is shown in Formula (2), which is as follows:ectively .Five relatively flat sites were selected for each CSF/PLA composite, and each site was tested for hydrophilic contact with a contact angle meter. The hydrophilic contact angle at the 30 s was taken as the test value. The average value is the hydrophilic contact angle test value of the material.