This paper focuses on the detailed design issues of a peculiar quadrature reduction method named system stiffness matrix diagonalization, whose key technology is the design and application of quadrature compensation patterns. For bulk silicon micro-gyroscopes, a complete design and application case was presented. The compensation principle was described first. In the mechanical design, four types of basic structure units were presented to obtain the basic compensation function. A novel layout design was proposed to eliminate the additional disturbing static forces and torques. Parameter optimization was carried out to maximize the available compensation capability in a limited layout area. Two types of voltage loading methods were presented. Their influences on the sense mode dynamics were analyzed. The proposed design was applied on a dual-mass silicon micro-gyroscope developed in our laboratory. The theoretical compensation capability of a quadrature equivalent angular rate no more than 412 °/s was designed. In experiments, an actual quadrature equivalent angular rate of 357 °/s was compensated successfully. The actual compensation voltages were a little larger than the theoretical ones. The correctness of the design and the theoretical analyses was verified. They can be commonly used in planar linear vibratory silicon micro-gyroscopes for quadrature compensation purpose. They are nowadays widely used in commercial and military fields, such as consumer electronics, automobile industry, aerospace navigation, weapons and military supplies, etc.Silicon micro-gyroscopes have achieved rapid development in the past several decades. In contrast with their traditional counterparts, silicon micro-gyroscopes have the advantages of small size, reduced power consumption and batch fabrication, The silicon micro-gyroscope is a kind of Coriolis vibratory gyroscope. It commonly works based on the Coriolis-effect-induced energy transmission between two orthogonal vibration modes, namely drive mode and sense mode. Generally, a Coriolis mass is actuated into resonant vibration with constant amplitude in the drive direction. When an angular rate is applied, a Coriolis-effect-induced Coriolis force will appear in the sense direction, which is proportional to the drive-mode velocity as well as the value of the input angular rate. Actuated by this Coriolis force, a Coriolis vibration response exists in the sense direction. Therefore, through the detection of the sense-mode position, the value of the input angular rate can be obtained.However, due to non-ideal factors such as fabrication imperfections, other coupling mechanisms also exist between these two vibration modes, introducing bias into the gyroscope output. The commonly seen coupling mechanisms are elastic coupling, viscous coupling and electrostatic coupling, among which elastic coupling is the largest in magnitude most of the time. Elastic coupling is mainly caused by the anisoelasticity either existing in the suspension elements themselves or introduced by fabrication imperfections, that is, an off-diagonal coupling stiffness often exists in the mechanical stiffness matrix of the micro-gyroscope structure. Because of this mechanical coupling stiffness, a quadrature force will appear in the sense direction, which is proportional to the drive-mode position. Therefore, quadrature vibration response also exists in the sense direction and will mix into the gyroscope output if it is not thoroughly distinguished from the Coriolis vibration response. In comparison with the amplitude of the Coriolis response, that of the quadrature response is often considerably large. Hence, quadrature reduction has become one of the primary issues in the design of high-performance silicon micro-gyroscopes.(1)Synchronous demodulation. During signal processing, the Coriolis signal is distinguished from the quadrature signal through synchronous demodulation according to their 90° phase difference.(2)Targeted signal injection. A compensation signal with the same amplitude but opposite phase is generated and injected into the input of the detection circuits to counteract the quadrature signal.So far various quadrature reduction methods have been reported ,2. They (1)Mechanical stiffness matrix diagonalization. The off-diagonal entry of the mechanical stiffness matrix is eradicated by post-fabrication trimming, e.g., laser trimming, on each individual microstructure. Once the mechanical stiffness matrix becomes diagonal, the quadrature vibration response no longer exists.(2)System stiffness matrix diagonalization. An electrostatic stiffness matrix is constructed with purposefully designed quadrature compensation patterns and properly applied DC voltages. The system stiffness matrix is then the sum of the naturally existed mechanical stiffness matrix and the artificially introduced electrostatic stiffness matrix. When the off-diagonal entry of the electrostatic stiffness matrix has the same value but opposite sign with that of the mechanical stiffness matrix, the system stiffness matrix would be diagonalized and the quadrature vibration response no longer exists.(3)Force feedback balancing. A balancing force that has the same value but opposite phase with the quadrature force is generated by the closed-loop force feedback circuits to cancel out the quadrature vibration response.In the movement correction category, there are mainly three specific methods:Obviously, among these quadrature reduction methods, the movement correction category is superior to the signal nulling category because it eliminates the quadrature vibration response at the source. Furthermore, among the three specific movement correction methods, system stiffness matrix diagonalization is superior to the other two. Compared with mechanical stiffness matrix diagonalization, it costs rather less and is much easier to control. Compared with force feedback balancing, it extracts the frequency and phase information mechanically from the drive-mode position, hence it avoids the need for precise frequency and phase control.The core idea of system stiffness matrix diagonalization is the modification of the system stiffness distribution, that is, a proper distribution of electrostatic stiffness is introduced to balance the non-ideal distribution of mechanical stiffness. This method has already been employed in vibrating ring or cup gyroscopes for a long time. In 1996, Clark first introduced its application in surface silicon micro-gyroscopes . HoweverThe rest of this paper is organized as follows: Section 2 describes the compensation principle of system stiffness matrix diagonalization based on the introduction of the arising mechanism and the characteristics of the quadrature vibration response. Section 3 discusses the mechanical design issues of system stiffness matrix diagonalization, including the classification of the basic structural units, the layout design of quadrature compensating patterns and the optimization of key structural parameters. Section 4 discusses the voltage loading methods of system stiffness matrix diagonalization. Two methods were proposed and their influences on the sense-mode dynamics were analyzed. Section 5 presents an actual application of the design theories on a dual-mass silicon micro-gyroscope developed in our laboratory. Section 6 provides the experimental results on a packaged silicon micro-gyroscope prototype. The correctness of the design and the theoretical analyses was verified. Section 7 concludes the whole paper.2.c represents the Coriolis mass; cx, cy represent the damping along X-axis and Y-axis; kx, ky represent the stiffness along X-axis and Y-axis; kxy and kyx are the mechanical coupling stiffness between drive and sense directions which bring about the quadrature vibration response; Fd is the driving force and Fc is the Coriolis force.Quadrature vibration response is mainly caused by the off-diagonal entry of the mechanical stiffness matrix . Considei.e., y ≪ x. Hence, the influence of kxy and y on the solution of x was ignored in the following analysis. We defined the quadrature force as Fq. Then the dynamic equation in drive direction and that in sense direction can be expressed as:Considering the common seen condition that the natural frequencies of the drive mode and the sense mode are mismatched to ensure a certain open-loop bandwidth, the mass position in sense direction would be much smaller than that in drive direction, c and the quadrature force Fq can be expressed as:z is the value of the input angular rate about Z-axis. The Coriolis mass is usually actuated into resonant vibration with constant amplitude in drive direction. Therefore, the driving force Fd and the corresponding solution of drive-mode position x can be expressed as:0 is the amplitude of the driving force, ωd is the natural frequency of the drive mode and Ad is the amplitude of the drive-mode position. In c is proportional to the drive-mode velocity and quadrature force Fq is proportional to the drive-mode position. With the same frequency they have a 90° phase difference with each other. With The Coriolis force Fc as Coriolis vibration response yc, and the sense-mode position caused by the quadrature force Fq as quadrature vibration response yq. With s is the natural frequency of the sense mode, Qs is the quality factor of the sense mode and φ is the phase shift, which can be expressed as:We defined the sense-mode position caused by the Coriolis force Fc and quadrature vibration response yq have the same relative relationship as that which exists between Coriolis force Fc and quadrature force Fq, that is, yc and yq still have the same frequency and a 90° phase difference with each other. It is obvious that though yc and yq are both affected by the quality factor and the natural frequencies, the relative relationship between them is independent of the sense-mode dynamics. Therefore, the quadrature equivalent angular rate Ωq of the quadrature vibration response yq can be expressed as:From KM, KE and KS denoting the mechanical, electrostatic and system stiffness matrix respectively, the compensation principle of system stiffness matrix diagonalization can be described as:ex, key represent the electrostatic stiffness along X-axis and Y-axis respectively; kexy, keyx are the electrostatic coupling stiffness between the drive and sense directions contributing to the quadrature compensation. When the electrostatic coupling stiffness has the same value but opposite sign as the mechanical coupling stiffness, the system stiffness matrix would be diagonalized and quadrature vibration response no longer exists.The key technology of system stiffness matrix diagonalization is the proper construction of an electrostatic stiffness matrix. With 1 and V2, are applied on Pad-1 and Pad-2 respectively. Considering the common seen condition that the mass position in sense direction is much smaller than the initial gap of the parallel plate capacitors, i.e., y ≪ d0, the electrostatic stiffness matrix provided by this quadrature compensation patterns and the DC voltages can be expressed as:0 is the permittivity of vacuum, h0 is the thickness of the structure and l0 is the initial overlap length of the parallel plate capacitors.The electrostatic stiffness matrix could be electromechanically provided by purposefully designed quadrature compensation patterns and properly applied DC voltages. A typical example of quadrature compensation patterns used in surface silicon micro-gyroscopes are shown in It can be seen from 3.0, i.e., pd0, the anchoring area of all pads would be continuous. Though the electrostatic coupling stiffness provided by a single set of comb capacitors is a little smaller than that of parallel plate capacitors, the available number of the comb capacitor sets would be much more than that of the parallel plate capacitor sets in a limited layout area. Therefore, in bulk silicon micro-gyroscopes, quadrature compensation patterns which consist of comb capacitors can provide a better overall compensation capability than those consist of parallel plate capacitors. In the following discussion, we focus on the design issues of quadrature compensation patterns which consist of comb capacitors in bulk silicon micro-gyroscopes.The quadrature compensation patterns illustrated in 3.1.dc is the applied DC voltage. For the convenience of analysis, we defined three structural constants, α, β and γ, as follows:According to the relative position between the Coriolis mass and the stationary pads, there are four types of basic structural units in the quadrature compensation patterns. The structural schematics of them along with the electrostatic forces they generated in drive and sense directions are shown in FE denotes the electrostatic force vector, Fex, Fey represent the electrostatic force in drive and in sense direction respectively, n is the number of the comb capacitor sets, l0 is the initial overlap length of the combs and x is the drive-mode position of the Coriolis mass.Identified by four different subscripts, A, B, C and D, the electrostatic forces and electrostatic stiffness matrixes provided by the four types of basic structural units can be expressed as:0 and the dynamic force is proportional to the drive-mode position x. The orientations of the static forces are determined by the relative position between the Coriolis mass and the stationary pads. They are always attractive and tend to increase the capacitor size. The needed electrostatic coupling stiffness is provided by the dynamic forces. In the four types of basic structural units, Type-A and Type-D generate dynamic forces with a negative orientation in sense direction, providing positive electrostatic coupling stiffness. Type-B and Type-C generate dynamic forces with a positive orientation in sense direction, providing negative electrostatic coupling stiffness.From For an actual silicon micro-gyroscope, the sign of the mechanical coupling stiffness is uncertain due to the uncertainty of the fabrication imperfections. Therefore, to ensure the feasibility of quadrature compensation, at least two types of basic structural units providing electrostatic coupling stiffness with opposite signs are needed in the mechanical design of quadrature compensation patterns.3.2.i.e., the layout design of quadrature compensation patterns.As discussed previously, among the electrostatic forces generated by the basic structural units, besides the dynamic forces which provide the needed electrostatic coupling stiffness, additional static forces also exist in the drive and sense directions. Moreover, with a lever arm between different basic structural units distributed at different places of the planar micro-gyroscope structure, additional static torques may also exist. These additional static forces and torques would cause a disturbance on the movement of the Coriolis mass. They can be cancelled out by a proper arrangement of the basic structural units on the available layout area of the Coriolis mass, md sets of basic structural units, including Type-A, Type-B, Type-C and Type-D, are designed in the middle part of the Coriolis mass. All the moveable combs in the four types of basic structural units are axisymmetric. Similarly, nsd sets of basic structural units are designed at the two side parts of the Coriolis mass with all the moveable combs axisymmetric. The basic structural units Type-A and Type-D providing positive electrostatic coupling stiffness are applied with a DC voltage V1. The basic structural units Type-B and Type-C providing negative electrostatic coupling stiffness are applied with another DC voltage V2.A novel layout design we propose to cancel out the additional static forces and torques is shown in As shown in In the sense direction, as discussed previously, each electrostatic force can be seen as the combination of a static force and a dynamic force. In our analyses, the forces with the same action line in sense direction were added up to get a resultant force. We use the subscripts “sta” and “dyn” to identify the static and dynamic forces, subscripts “md” and “sd” to identify the locations of the basic structural units. As shown in sta_mdAB and Fsta_mdCD are zero and that of Fsta_sdAB and Fsta_sdCD are also zero. Hence there is no net static force. However, with a lever arm Lmd exists between Fsta_mdAB and Fsta_mdCD, a static torque Mmd turns up. Similarly, with a lever arm Lsd exists between Fsta_sdAB and Fsta_sdCD, a static torque Msd turns up. These two static torques can be expressed as:It is obvious that the sum of FAiming to eliminating these static torques, in our layout design, the number of the capacitor sets and the lever arms were designed to follow the following rule:md and Msd would cancel out with each other. Hence there is no net static torque as well. With md and Msd can be explained as follows:In this condition, MIn conclusion, with the proposed layout design of quadrature compensation patterns, the additional static forces and torques generated by the basic structural units can be cancelled out. Only the useful dynamic forces providing electrostatic coupling stiffness exist in the sense direction.The four dynamic resultant forces in sense direction can be expressed as:dyn and the electrostatic coupling stiffness keyx it provides can be expressed as:The net dynamic force Feyx can be obtained as:With q, the needed value of the DC voltages for quadrature compensation would be:With 1 and V2 is determined by the phase information of the quadrature vibration response.The needed relative size of VY-axis is inevitably introduced as long as the comb capacitors have an initial overlap length. The negative electrostatic stiffness key introduced by the novel layout design of quadrature compensation patterns shown in In addition, it can be found From ey can be obtained as:With 3.3.Obviously, the quadrature compensation capability is determined by the value of the electrostatic coupling stiffness provided by the quadrature compensation patterns and the applied DC voltages. Considering the power consumption of a silicon micro-gyroscope, the available values of the DC voltages are generally limited. Therefore, an optimization of the structural parameters is necessary to improve the quadrature compensation capability.0, the initial gap d0, the set numbers nmd, nsd and the gap ratio p (p > 1). Among these parameters, h0 and d0 are often chosen according to the bulk silicon micro-fabrication process. Increasing the gap ratio p can increase the electrostatic coupling stiffness provided by a single set of basic structure unit. However, it would decrease the set numbers nmd, nsd at the same time. Therefore, a proper design of the gap ratio p is crucial in parameter optimization.From md, Lsd and the set numbers nmd, nsd follow the design rule shown in In our layout design shown in qy, and the comb width in the basic structural units as w. Then the available set numbers would be:We defined the available length of the Coriolis mass along Y-axis which can be used for the design of quadrature compensation patterns as LWith 0 and d0 have constant values, keyx and p would have the following relationship:eyx can be obtained with a certain value of p.Therefore, when w, h0 is most commonly chosen as 4 μm. Then with several different values of the comb width w, the influences of the gap ratio p on the value of the variable η are shown in eyx can be maximized within a limited available length Lqy. When the comb width w was chosen as 4, 6 and 8 μm respectively, the optimal values of p would be 2.36, 2.49 and 2.61.Considering the bulk silicon micro-fabrication process adopted in our laboratory, the initial gap d4.q with the variation of V1 and V2. The main influence of quadrature compensation on the dynamic equation in sense direction is the introduction of an electrostatic coupling stiffness keyx and a negative electrostatic stiffness key. With no input angular rate, the dynamic equation in sense direction during quadrature compensation can be expressed as:In the process of quadrature compensation, the regulation of the DC voltages relies on the change tendency of the quadrature vibration response yq in the process of quadrature compensation can be expressed as:With 1 and Group-2 is applied with DC voltage V2. Generally, there are two voltage loading methods:(1)1 or V2, the amplitude of the quadrature vibration response was reduced to zero. The voltage loading group was chosen manually. Hence this method is suitable for off-line compensation.Single group loading. The sign of the mechanical coupling stiffness was firstly obtained through measuring the phase of the quadrature vibration response. Then according to the needed sign of the electrostatic coupling stiffness, positive or negative, only one group of the patterns, Group-1 or Group-2, was applied with a DC voltage. Through the regulation of this DC voltage, V(2)1 and V2. The sign of the electrostatic coupling stiffness, positive or negative, was decided by the relative size of V1 and V2. Through the regulation of |V12 − V22|, the amplitude of the quadrature vibration response was reduced to zero. Commonly, these two DC voltages were chosen as follows:Double group loading. Both groups of the patterns, Group-1 and Group-2, were applied with DC voltages, Vwhere VD is the preset bias voltage and Vq is the regulation voltage. With a constant VD, the regulation of Vq can change both the sign and the value of the electrostatic coupling stiffness. Hence this method is suitable for on-line compensation.For the convenience of analysis, we named the upper half of the quadrature compensation patterns shown in q| and the applied DC voltage V1 can be expressed as:When single group loading was employed, assuming that the mechanical coupling stiffness was negative, with D was preset. With q| and the regulation voltage Vq can be expressed as:When double group loading was employed, a constant bias voltage Vq| has a non-linear relationship with V1. The natural frequency of the sense mode was directly affected by V1. It would change a lot in the process of quadrature compensation. From D ≫ Vq was met, the influence of Vq on the natural frequency of the sense mode can be alleviated greatly and |yq| would have an approximately linear relationship with Vq. Therefore, when a smaller change of the natural frequency or a linear relationship between the output and input signals was preferred in the process of quadrature compensation, double group loading would be superior to single group loading.From q| would also be nonzero and the quadrature vibration response would be in the state of overcompensation.No matter which voltage loading method was employed, the need quadrature compensation voltage is the one that makes the amplitude of the quadrature vibration response |yq| to be zero. When a voltage smaller than that one was applied, |yq| would be nonzero and the quadrature vibration response would be in the state of undercompensation. When a voltage larger than that one was applied, |y5.The proposed design of quadrature compensation patterns shown in q no more than 412 °/s can be realized theoretically by the proposed design.The main design parameters are shown in 6.The dual-mass silicon micro-gyroscopes were vacuum sealed with ceramic packages after micro-fabrication. Off-line quadrature compensation was carried out on a silicon micro-gyroscope prototype to verify the correctness of the previous design and analyses.q. Hence, after measuring the amplitude of the sense-mode position, with q was estimated. Then with In the process of off-line quadrature compensation, the drive circuit worked normally. That is, in the drive direction, the two Coriolis mass were actuated into resonant vibration in anti-phase mode with the same constant amplitude. With no input angular rate, the drive-mode velocity signal and the sense-mode position signal were both extracted. In this condition, the sense-mode position signal was dominated by the quadrature vibration response yq was estimated to be 357 °/s. The sign of its mechanical coupling stiffness was found to be negative. Therefore, with 12 − V22| ≈ 118.6 V2. When the voltage loading method of single group loading was employed, Group-1 was needed to be applied with a DC voltage of V1 ≈ 10.89 V theoretically. When the voltage loading method of double group loading was employed, with a preset bias voltage of VD = 10 V applied on both Group-1 and Group-2, the needed regulation voltage would be Vq ≈ + 2.97 V theoretically. The two voltage loading methods were both carried out and the experimental data of the relationships between the output amplitude of the sense-mode position signal and the applied DC voltages were obtained. At the same time, the theoretical curves of the relationships between the amplitude of the quadrature vibration response and the applied DC voltages were calculated from The experimental results of a silicon micro-gyroscope prototype showing quite large quadrature vibration response are presented here. The natural frequency of its drive mode is about 3.8 kHz. The value of its quadrature equivalent angular rate Ω1. The actually needed DC voltage for quadrature compensation was V1 ≈ 12 V, which is a little larger than the theoretical value.When the voltage loading method of single group loading was employed, the comparison of the experimental data and the theoretical curve was shown in q. The actually needed DC voltage for quadrature compensation was Vq ≈ 3 V, which is also a little larger than the theoretical value.When the voltage loading method of double group loading was employed, the comparison of the experimental data and the theoretical curve was shown in The drive-mode velocity and sense-mode position signals before off-line quadrature compensation are shown in It is obvious that the sense-mode position signal has the same frequency as the drive-mode velocity signal. Before off-line quadrature compensation, it was dominated by the quadrature vibration response and had an approximately 90° phase lag with the drive-mode velocity signal. After off-line quadrature compensation, the quadrature vibration response was cancelled out. However, it could be seen that a residual sense-mode position signal still existed. Its amplitude was about 100 mV in the tested silicon micro-gyroscope prototype. This residual signal had the same phase with the drive-mode velocity signal and is commonly named as in-phase error. It is mainly caused by other mechanisms, e.g. the viscous coupling between drive mode and sense mode.7.Quadrature reduction is one of the primary issues in the design of high-performance silicon micro-gyroscopes. Among the various quadrature reduction methods, system stiffness matrix diagonalization has its peculiar advantages. In this method, with purposefully designed quadrature compensation patterns and properly applied DC voltages, an electrostatic coupling stiffness is electromechanically constructed to cancel out the mechanical coupling stiffness which is the main cause of the quadrature vibration response. This paper focus on the detailed design issues of this method and presents a complete design and application case.For bulk silicon micro-gyroscopes, the quadrature compensation patterns which consist of comb capacitors are more suitable than those consisting of parallel plate capacitors because they provide a better overall compensation capability. There are four types of basic structural units in the mechanical design of quadrature compensation patterns. With an applied DC voltage, they can generate negative or positive dynamic electrostatic forces in sense direction. These dynamic forces are proportional to the drive-mode position, hence the needed positive or negative electrostatic coupling stiffness can be provided. Besides the useful dynamic forces, the basic structure units also generate additional static forces and torques which may cause a disturbance. A novel layout design was proposed to solve this problem. The basic structural units distributed at different places can provide same-directional dynamic forces, opposite-directional static forces and opposite-directional static torques. The opposite-directional static forces were designed to have the same absolute value. Hence no net static force exists. The opposite-directional static torques were also made to have the same absolute value. Hence no net static torque exists. When the available values of the applied DC voltages are limited, an optimization of the structural parameters, especially the gap ratio, can help improving the quadrature compensation capability.In the process of quadrature compensation, there are two voltage loading methods: single group loading and double group loading. Single group loading is more suitable for off-line quadrature compensation. When it is employed, the quadrature signal has a non-linear relationship with the compensation voltage in the compensation process. The natural frequency of the sense mode is directly affected during the regulation of the compensation voltage. Double group loading is more suitable for on-line quadrature compensation. When it is employed, the quadrature signal has an approximately linear relationship with the compensation voltage in the compensation process. The influence of the compensation voltage regulation on the natural frequency of the sense mode can be alleviated.The proposed design of quadrature compensation patterns was applied on an actual dual-mass silicon micro-gyroscope developed in our laboratory. With the DC voltage applied on a single pattern group no more than 12 V, the theoretical compensation capability of a quadrature equivalent angular rate no more than 412 °/s was designed. In the experiments carried out on a packaged micro-gyroscope prototype, an actual quadrature equivalent angular rate of 357 °/s was compensated. The values of the actual compensation voltages were a little larger than the theoretical ones. The correctness of the design and the theoretical analyses was verified. They can be commonly used in planar linear vibratory silicon micro-gyroscopes for quadrature compensation purpose.


In the current year, the Brazilian Society of Urology celebrates its 90th year of continuous activity and the official scientific journal of the society “International Brazilian Journal of Urology” will present several important modifications throughout this festive year.The electronic Int Braz J Urol page (www.brazjurol.com.br) was updated and became more interactive and friendly, facilitating the access to all information. From now on, it is possible to directly access all articles published from the first publication of the Brazilian Journal of Urology (the former name of our magazine), providing instant consultation. Visit the new webpage in www.intbrazjurol.com.brFinally, Int Braz J Urol will become part of Pubmed Central, the free access to the digital collection of the National Center for Biotechnology Information (NCBI). With this achievement, our Journal will be more widespread in the international scientific community.Read Int Braz J Urol, publish at the Int Braz J Urol, quote the good papers of Int Braz J Urol!Sidney Glina, MD, PhDEditor-In-ChiefInternacional Braz J Urol


Individual joint deviations are often identified in the analysis of cerebral palsy (CP) gait. However, knowledge is limited as to how these deviations affect the control of the locomotor system as a whole when striving to meet the demands of walking. The current study aimed to bridge the gap by describing the control of the locomotor system in children with diplegic CP in terms of their leg stiffness, both skeletal and muscular components, and associated joint stiffness during gait. Twelve children with spastic diplegia CP and 12 healthy controls walked at a self-selected pace in a gait laboratory while their kinematic and forceplate data were measured and analyzed during loading response, mid-stance, terminal stance and pre-swing. For calculating the leg stiffness, each of the lower limbs was modeled as a non-linear spring, connecting the hip joint center and the corresponding center of pressure, with varying stiffness that was calculated as the slope (gradient) of the axial force vs. the deformation curve. The leg stiffness was further decomposed into skeletal and muscular components considering the alignment of the lower limb. The ankle, knee and hip of the limb were modeled as revolute joints with torsional springs whose stiffness was calculated as the slope of the moment vs. the angle curve of the joint. Independent t-tests were performed for between-group comparisons of all the variables. The CP group significantly decreased the leg stiffness but increased the joint stiffness during stance phase, except during terminal stance where the leg stiffness was increased. They appeared to rely more on muscular contributions to achieve the required leg stiffness, increasing the muscular demands in maintaining the body posture against collapse. Leg stiffness plays a critical role in modulating the kinematics and kinetics of the locomotor system during gait in the diplegic CP. Cerebral palsy (CP) is an irreversible and non-progressive disorder caused by brain injury before, during or shortly after birth . During Human walking is achieved by a highly complex interaction of the force-bearing structures of the musculoskeletal system. While sophisticated models of the system have been used to provide insights into the gait mechanics in children with CP , a simplet al. (2000) [Leg stiffness incorporates information of both the kinematics and kinetics of the lower limb joints because both the leg length and the GRF are affected by the joint angles and moments at any instance of gait , 9, 12. . (2000) , the leg. (2000) . Study o. (2000)  first de. (2000) . Applica. (2000) . HoweverThe purpose of this study was to investigate the leg and joint stiffness, the contributions of skeletal and muscular components, and the associated joint kinematics and kinetics in children with spastic diplegia CP during level walking. It was hoped that the results will provide baseline data on the stiffness control of the lower limb as a whole in healthy children and in children with diplegic CP, which would be helpful in future assessment of the effects of the pathology and the efficacy of relevant treatment methods.This study was approved by the Institutional Research Board of National Taiwan University Hospital (Permit Number: 201305084RINC). All the subjects and their parents/guardians were informed of the procedure and provided written informed consent prior to the study, including enrolment and data collection.a priori power analysis based on pilot results on leg and joint stiffness using GPOWER [Twelve children with spastic diplegia cerebral palsy , and twelve age-matched healthy controls  participated in this study. The CP group was graded 1–3 in the Gross Motor Function Classification System (GMFCS) with slightly limited passive range of motion (ROM) of the hip and ankle, moderate to normal muscle strength and mild to normal muscle tone . Howeverg GPOWER  determinEach subject walked at a self-selected pace on an 8-meter walkway. Each subject wore 39 retroreflective markers for tracking the motions of the body segments  using a With the measured GRF and kinematic data, angular motions and internal moments at the lower limb joints were calculated using inverse dynamics analysis . Each boFe(t)), is defined as the component of the GRF of the limb along the line joining the COP and the hip joint center. The length of the leg spring is defined as the distance between the hip joint center and the COP, and is called the effective leg length (Le(t)). Therefore, the leg stiffness, Kl(t), at time t could be calculated as the gradient of the Fe(t) vs. Le(t) curve as follows  of the force vs. deformation curve. The force applied to the leg spring, called effective GRF ( follows :Kl(t)=dKa(t)), knee (Kk(t)), and hip (Kh(t)) at time t were calculated as the slope of the moment (M(t)) vs. angle (θ(t)) curve of the joint as follows:Similarly, the joint stiffness of the ankle (Fe(t), Le(t), (θ(t)) and M(t) were low-pass filtered using a fourth-order bi-directional Butterworth filter with a cut-off frequency of 10 Hz [Ks,l (t) is the skeletal component in Kl (t); Km,l (t) is the muscular component in Kl (t); Ks(t) is the skeletal stiffness; and Km(t) is the muscular stiffness (t) = 0) lower limb would have the leg stiffness provided solely by the skeletal component. As the angle (φ) increases, the contribution of the muscular component to the leg stiffness increases with the increasing lever arm length available to the effective GRF at the knee =Ktiffness . Based ocos2φ(t);Km,l(t)=)cosφ(t);where φ and double limb support (DLS), using the COP and GRF data measured for each limb. The results were time-averaged over the sub-phases of the stance phase, namely loading response , mid-stance, terminal stance and pre-swing  for each limb. The data from both limbs were then averaged for each trial for each subject. These gait phases were determined using the forceplate and marker data. Temporal-spatial parameters, namely walking speed, stride length normalized to leg length, stride time, cadence and step width, were also obtained. For data presentation, the curves of each angle and moment component at each joint from all the subjects were ensemble-averaged to obtain the means and standard deviations during the gait cycle for each subject group.For each of the calculated variables independent t-tests were performed to compare each of the time-averaged values over the sub-phases between the CP and control groups. The effect of gait speed on the between-group comparisons in all stiffness-related variables was tested using analysis of covariance (ANCOVA) with the gait speed as a confounding factor. All significance levels were set at α = 0.05. All the statistical analyses were performed using SPSS version 20.0 .Compared to the healthy controls, the CP group showed significantly increased stride time and step width, but significantly reduced gait speed and cadence . HoweverCompared to the control group, the CP group showed significantly increased flexion at the hip and knee during most of the stance phase, and increased ankle plantarflexion during terminal stance and the pre-swing phase . The CP Compared with the healthy controls, the CP group showed significantly reduced leg stiffness during loading response and mid-stance, with increased joint stiffness at the hip, knee and ankle . During Compared with the healthy controls, the children with CP walked with a significantly increased muscular component but reduced skeletal component of the leg stiffness during loading response and mid-stance, giving a significantly increased ratio of the muscular to skeletal components . The musThe current study was the first attempt to quantify the leg stiffness and the associated skeletal and muscular components, as well as joint stiffness, during stance phase of gait in children with spastic diplegia CP. In the literature, some studies calculated the leg stiffness as the ratio between the peak GRF and maximum displacement , 27, givDuring loading response or initial DLS, children with CP showed a reduced leg stiffness even though the stiffness of individual joints was increased compared to the control group. This appeared to be a result of the more flexed posture of the lower limbs  while acDuring the pre-swing phase, the reduced leg stiffness but increased joint stiffness in the CP group suggests that the subjects may have a compromised ability for providing a pushing-off force for the subsequent swing. Since the body weight is transferring from the stance limb to the contralateral limb, the leg stiffness and the walking stability during this period did not have to be as high as that during weight acceptance. However, instability in the control of the stance limb during the unloading phase may affect the smooth transfer of the body weight, leading to an increased joint stiffness and muscular component of leg stiffness. The reduced leg stiffness but increased joint stiffness in both limbs during DLS suggest that the children with diplegic CP had altered coordination of the transfer of the body weight and modulation of the leg and joint stiffness between the limbs, leading to a higher risk of imbalance or collapse during weight transfer.During mid-stance, the objective was to ensure smooth progression of the body over the stationary foot, which had to be based on necessary stability of the lower limb. Following loading response, the more flexed posture of the lower limbs during this period also contributed to the reduced leg stiffness in the children with CP, requiring increased muscular components and joint stiffness. This suggests that when subjected to a sudden increase in the GRF, e.g., extra force needed to recover balance, the risk of collapse in these children may increase.During terminal stance, the CP group increased the leg stiffness primarily via increased knee joint stiffness, presumably to prepare for transferring the body weight to the contralateral limb. This would also be helpful for preventing an undesired reduction in toe clearance of the contralateral limb owing to increased shortening of the ipsilateral leg length. This is particularly important in preventing stumbling as their ankle dorsiflexion angles were reduced . It appeThe CP group appeared to rely more on muscular contributions to achieve the required leg stiffness. The more flexed hip and knee increased the lever-arm length of the GRF vector at the joint centers, and thus the joint moments. This also increased the contribution of the musculature system to the leg stiffness but decreased that of the skeletal system . The incWith a more flexed posture during the stance phase, the CP group had to sustain increased loads at the individual joints, as well as increased joint stiffness. However, these changes did not maintain the leg stiffness within the normal range. This appeared to be related to the inability to modulate the leg stiffness to compensate for the symptoms of the neurological changes and the muscle weakness during gait . DecreasThe leg stiffness and related variables proposed in the current study provide a measure for assessing the control of the lower extremities as a whole in supporting the body against collapse during gait, which would be very helpful in the assessment of the effects of the pathology and the efficacy of relevant treatment methods in a clinical setting. This information is not readily available from the individual joint angular and moment data, nor from the EMG data of the relevant muscles obtained using traditional gait analysis. It is thus suggested that the stiffness-related variables should be included in future clinical gait analysis for a more complete assessment of gait in children with CP. As a first step, the current study demonstrated that these variables successfully distinguish the control of the lower limb as a whole between diplegic CP and healthy subjects, providing baseline data for these subject groups. On the other hand, while leg stiffness-related variables integrate both force and displacement information , how theLeg stiffness plays a critical role in modulating the kinematics and kinetics of the locomotor system during gait in diplegic CP. The CP group significantly decreased the leg stiffness but increased the joint stiffness during stance phase, except during terminal stance where the leg stiffness was increased. They appeared to rely more on muscular contributions to achieve the required leg stiffness, increasing the muscular demands in maintaining the body posture against collapse. Therefore, therapeutic decision-making and planning should consider not only the mechanics of individual joints, but also the leg stiffness control. The stiffness-related variables should be included in future clinical gait analysis for a more complete assessment of gait in children with CP.S1 Dataset(CSV)Click here for additional data file.


Giant cell tumour in the metatarsal of a skeletally immatureperson is uncommon. Adequate surgical resection in thisregion can be difficult to achieve as there is little spacebetween the rays of the foot. Furthermore, there is thechallenge of restoring the metatarsophalangeal articulationafter resection of the tumor. We describe a technique usingnon-vascularised fibular graft for reconstructing the 2ndmetatarsal after en-bloc resection for giant cell tumour in a 14year old adolescent female.Metatarsal, Giant cell tumour, Non-vascularised fibulargraft, En-bloc resection Giant cell tumour (GCT) remains an enigmatic bone tumourdespite being extensively studied. The World HealthOrganization has classified GCT as “an aggressive,potentially malignant lesion”. The histology does not predictthe clinical outcome, and different anatomical sites presentwith different management problems and difficultiesGCT is estimated to represent 15% of benign and 3% to 8%of all bone tumoursA 14 year old Chinese girl presented with swelling over theright foot , first nRadiograph of the right foot  revealedThe tumour was excised en-bloc and - a marginal surgicalexcision as defined by Enneking was achieved. The 2ndmetatarsal was reconstructed with a non-vascularised fibulargraft and fixed with a small dynamic compression plate, thebase of the 2nd metatarsal bone was preserved withoutviolating the 2nd tarso-mtatarsal joint (TMT) joint. The distalpart of the fibular graft was shaped to resemble the distalmetatarsal condyle to fit the articulation with the proximalphalanx of the 2nd toe. The resected length was measured andrestored to ensure correct soft tissue tension in themetatarsophalangeal joint. Intra-operatively the joint wasstable.The postoperative period was uneventful and the patient wasdischarged on the 10th postoperative day. A cast was appliedfor 3 months to protect the graft and fixation. She was allowedpartial weight bearing after 3 months and full weight bearingafter 6 months. At the last review at 24 months the graft hadincorporated and united . The metHistopathological examination confirmed the diagnosis ofhigh grade GCT of bone with complete resection of tumour.Treatment of choice in most GCT involves curettage and bonegrafting with or without the use of treatment adjuvants likepost-curettage application of liquid nitrogen,methylmethacrylate, phenol, hydrogen peroxide and alcohol.Preservation of limb and motion in the affected joint is achallenge as GCT is commonly found in the epiphysis andoften invades the subchondral bone and the adjacent joint.This patient presented with a Campanacci Grade 3 tumour. Ingrade 3 tumours the cortex is often breached or the outercortical shell too thinned out to make curettage possible. Enblocresection is the treatment of choice.There is very little written about the reconstruction of themetatarsal after excision for GCT. The first and fifthmetatarsals are vital to maintaining the medial and laterallongitudinal arches of the foot and it is therefore important toreconstruct these structures when limb preservation iscontemplated to ensure good functional results. The decision toreconstruct the other metatarsals must be carefully consideredagainst donor site morbidity and technical difficulties.Treatment for GCT of the metatarsals may vary from resectionaloneWe describe a method which preserves metatarsophalangealjoint motion. The fibular graft can be easily shaped toresemble a metatarsal condyle with a burr. It is vital to restorethe resected length to ensure joint stability. The graft is fixedwith a dynamic compression plate.Although this is a single report of surgical reconstruction withnon-vascularised fibular graft after excision of giant celltumour of the metatarsal, the early outcome has been quiteencouraging. We feel that this technique may be a promisingsurgical option in the treatment of this difficult condition.


The affiliation for the authors Na Shang, Qian Li, and Tzu-Ping Ko are incorrect. Na Shang and Qian Li are affiliated only with Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China. Tzu-Ping Ko is affiliated only with the Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.


The gastro intestinal transit behavior of the formulation was determined by gamma scintigraphy, using 99mTc as a marker in healthy rabbits. The amount of radioactive tracer released from the labelled tablets was minimal when the tablets were in the stomach, whereas it increased as tablets reached to intestine. Thus, in-vitro and in-vivo drug release studies of starch-acrylate graft copolymers proved their controlled release behavior with preferential delivery into alkaline pH environment.The present investigation deals with the development of controlled release tablets of salbutamol sulphate using graft copolymers (St-g-PMMA and Ast-g-PMMA) of starch and acetylated starch. Drug excipient compatibility was spectroscopically analyzed via FT-IR, which confirmed no interaction between drug and other excipients. Formulations were evaluated for physical characteristics like hardness, friability, weight variations, drug release and drug content analysis which satisfies all the pharmacopoeial requirement of tablet dosage form. Release rate of a model drug from formulated matrix tablets were studied at two different pH namely 1.2 and 6.8, spectrophotometrically. Drug release from the tablets of graft copolymer matrices is profoundly pH-dependent and showed a reduced release rate under acidic conditions as compared to the alkaline conditions. Study of release mechanism by Korsmeyer’s model with n values between 0.61-0.67, proved that release was governed by both diffusion and erosion. In comparison to starch and acetylated starch matrix formulations, pharmacokinetic parameters of graft copolymers matrix formulations showed a significant decrease in C Controlled release technology has rapidly emerged over the past few decades as a new interdisciplinary science that offers novel approaches for delivery of bioactive agents into systemic circulation at a predetermined rate, achievement of optimum therapeutic responses, prolonged efficacy and decreased toxicity . Salbutain-vitro dissolution, pharmacokinetics and gamma scintigraphic evaluation in rabbits upon oral administration. For optimized formulations, release kinetics and drug release mechanism were evaluated by fitting dissolution profile in various kinetic models viz. zero order, first order, Higuchi and Korsmeyer-Peppas models.In our previous research paper, we reported the synthesis and characterization of pH sensitive acetylated starch-acrylate graft copolymer  and to eMaterialsLtd., India. Hydroxypropyl Methylcellulose K100M (HPMC K100M) was procured from Colorcon Asia Pvt. Ltd. and solvents of analytical grade were supplied by Merck Ltd., Germany. Gift sample of salbutamol sulphate was received from FDC Ltd., Mumbai, India. 99mTc was provided as a gift sample from Spect Lab Nuclear Medicine Services, Pune, India.Maize starch was procured from Meru Chem Pvt. Synthesis of acetylated starch and graft copolymers [Starch grafted poly (methyl methacrylate) (St-g-PMMA) & Acetylated starch grafted poly (methyl methacrylate) (Ast-g-PMMA)]Acetylated starch and graft copolymers were synthesized and characterized by the methods previously described in our recent published paper . Here onPreparation of blendsHPMC K100M polymer was chosen for comparison with graft copolymers for its controlled release properties. Various combinations were selected and blend was prepared for salbutamol sulphate using starch/acetylated starch/St-g-PMMA/Ast-g-PMMA/HPMC K100M, spay dried lactose and magnesium stearate. All ingredients were blended in Conta blender until a homogenous mixture was obtained with batch formula shown in Preformulation studyDrug–excipient compatibility study by FT-IR spectroscopyFT-IR analysis was performed using physical mixture (1:1 w/w) of salbutamol sulphate with starch/acetylated starch/St-g-PMMA/Ast-g-PMMA.Angle of reposeAngle of repose is an indicator of flowability of the material and it was determined by the fixed funnel and free standing cone method . In this-1 (h/r)θ= tanFormulation of tabletsTablets were prepared by direct compression method. The homogenous mixtures of different blends with suitable flow properties were shifted through #80 sieves, manually fed into the die and compressed in hydraulic press using a 8 mm flat faced beveled edge punch with the crushing force of 70-80 N. The average weight of tablets was 200 mg. Two hundred tablets of each batch were prepared and evaluated for various physicochemical parameters.Evaluation of tablets2) of prepared tablets was established using Monsanto hardness tester . Tablets friability was determined by Roche friabilator , and it was calculated as the percentage of weight loss in 20 tablets resulting from shock and attrition due to the revolution of plastic chamber operated for 4 minutes at 25 rpm.The physical testing on tablets of each batch was done after a relaxation period of at least 24 h. Weight variation test was performed on 20 individually weighed  tablets according to the official method of United State Pharmacopoeia (USP/NF-32). The thickness and diameter of 10 tablets were measured individually using Vernier Caliper  and an average value of thickness and diameter were calculated. The crushing strength  at 277 nm and the drug concentration was determined using constructed standard calibration curve, covering the drug solution concentration from 5.0-50.0 µg/mL.Radiolabelling the tablets with Technetium-99 (99mTc)99mTc) was chosen for radiolabelling of the tablets because of its short half-life of 6 h along with optimum electron emission. Diethylenetriamine-pentaacetic acid (DTPA) was used to provide the necessary 99mTc species for radiolabelling (99mTc-labeled DTPA was taken and 1 mg of stannous chloride dihydrate (1 mg/mL in 10% acetic acid) was added. The pH was adjusted to 7.5 using 0.5 M sodium bicarbonate solution and mixture was stirred for 5 minutes. Afterwards 4–7 µL of aqueous solutions of 99mTc-DTPA of known radioactivity (100 MBq) was instilled in to the drilled holes, avoiding contact with the surface of the tablets. The radioactive solution was left to diffuse for 15 minutes and then dried to assure uniform dispersion of radioactive material within the matrix. Ethanolic solutions of graft copolymers and lactose (9:1) were used to fully seal the drilled holes. The strength of radioactive label (MBq) was determined by CAPINTEC CRC-15R detector .Tablets were radiolabelled by drilling a small hole (1 mm) through the center of the tablet with an electric driller and introducing a radioactive solution . Technetabelling . In a viStability of radiolabelled tablets99mTc-labeled graft copolymer matrix tablets (F3 & F4) was tested in standard buffer solutions of pH 1.2, 6.8 and 7.4 in order to confirm that the activity would not leach out from the tablets during transit time of the formulation through GI tract. Dissolution tests for the release of the radioactive material was performed by placing radiolabelled tablets in USP type II paddle type dissolution apparatus containing different standard buffers solutions (900 mL), and it was operated at rotation speed of 50 rpm at 37 ± 0.5 °C for 6 h. At each 30 min interval, 5 mL of sample was withdrawn from dissolution vessel and withdrawn volume of dissolution media was replaced with fresh buffer solution. At the end of the dissolution testing, tablets were recovered from the dissolution medium and were further blotted using tissue paper. Activity of the test solutions was determined using a CRC-15R detector. The amount of radioactive strength remained in the tablets was estimated by dissolution testing. Stability of In-vitro drug release studies99mTc-DTPA labeled tablets and compared with unlabelled tablets to determine whether the labeling process affects the kinetics of drug release from the tablets. Drug release study was carried out using USP dissolution type II apparatus with paddle rotating at 50 rpm. Dissolution study was performed in simulated gastric fluid (0.1N HCl) and in simulated intestinal fluid (PBS pH 6.8) for 14 h. The temperature of dissolution medium (900 mL) was maintained at 37 ± 0.5ºC throughout the study. Tablets were placed in different baskets. At a predetermined time intervals of 0, 1, 2, 3, 4, 5, 6, 8, 10, 12 and 14 h, 5 mL sample was withdrawn using a syringe fitted with 0.45 μm filter, and withdrawn volume was replaced with the fresh medium to maintain sink condition. Samples were analyzed at 277 nm, using UV-visible spectrophotometer. The corresponding cumulative percent of drug released was determined using the constructed standard calibration curve at this wavelength covering the range for the assay. Mean release of three tablets was used to evaluate the drug release for each of the formulations (Drug release studies were conducted on ulations .Kinetics of drug releasein-vitro release profiles were correlated with various kinetic models like zero order (cumulative amount of drug released vs. time), first order (log cumulative percentage of drug remaining vs. time), Higuchi model (cumulative percentage of drug released vs. square root of time) and Korsmeyer-Peppas (log cumulative percentage of drug released vs. log time) release equations.To study the mechanism of drug release from the optimized formulation of matrix tablets, In-vivo Pharmacokinetic studies on rabbitsBio-analytical method development 18) with length 250 mm, internal diameter 4.6 mm (Phenomenex) and particle size 5 μm. Chloramphenicol was used as an internal standard. In 0.5 mL of plasma sample, 20 μL of internal standard (100 μg/mL) was added. Drug was extracted from plasma using 5 mL methanol. Mobile phase used for analysis contains acetonitrile, methanol and water in the ratio of 60:20:20 (v/v), and pH adjusted to 2.8 with orthophosphoric acid, with a flow rate of 0.5 mL/min. Sample of 20 μL was injected manually and chromatogram was recorded. Quantification of salbutamol sulphate was performed by plotting peak area ratio of SS to the internal standard as a function of its concentration.A high performance liquid chromatography system of Adept series CECIL CE 4201 with UV/Visible detector was used for analysis. The data were recorded by using the software “Power Stream”. The column used for separation was octadecylsilane  were compared with tablets prepared by native starch (F1), acetylated starch (F2), HPMC K100M (F5) and commercial sustained release tablets .viz. 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h. Samples were collected in micro centrifuge tubes containing 50 µL of 10% w/w of disodium EDTA as anticoagulant. The collected samples were immediately centrifuged at 10000 rpm for 10 minutes and plasma were separated and stored at –20 °C.Male albino rabbits weighing 2.5-3.0 kg were randomly selected for the bioavailability study. The animals were subdivided into six groups and each group was comprised of three rabbits. Each group received of the tested formulas namely F1, F2, F3, F4, F5 and Asthalin SA-8 mg. The animals were fasted over night before tablet administration and during the experiment all rabbits had free access to water. Tablets were put behind the tongue to avoid their destruction due to biting. Blood samples  were collected from the orbital sinus, before dosing (zero time) and at different intervals after dosing Statistical analysisstudent newmann-keuls multiple comparison test and p-values of <0.05 were considered to be statistically significant. Statistical significance was determined by one way ANOVA  . Significant differences between formulations were analyzed using In-vivo imaging and animal study protocol for gamma scintigraphy99mTc as a radioactivity marker. Male albino rabbits weighing 2.5-3.0 Kg were randomly selected for gamma scintigraphic study. Rabbits were divided into 3 groups, having 3 animals in each and were fasted for 12 h prior to the gamma scintigraphic study. Group-I received radiolabelled starch matrix tablet (F1), group-II & III received graft copolymer containing matrix tablets F3 and F4 respectively; followed by sufficient amount of drinking water. Animals were anaesthetized with diethyl ether and serial scintigraphic examination was done at 0.5, 1, 1.5, 2, 3, 5, 7 and 10 h to visualize the transit of tablets in GIT, using a large field view gamma camera equipped with a high-resolution, parallel-hole collimator. The 140 keV gamma rays emitted by 99mTc were imaged. Gamma images were recorded using an online computer system and analyzed to determine the distribution of radioactivity in the stomach, intestine and colonic region.The whole experiment was approved by “Institutional Animal Ethical Committee” according to the rules of “Committee for the Purpose of Control and Supervision of Experiments on Animals”  for the care and use of laboratory animals were strictly followed throughout the experiment. Gastrointestinal transit of tablets was obtained by imaging studies using Preformulation studyDrug–excipient compatibility study by FT-IR spectroscopy-1 corresponding to C-O stretching and at 1643 cm-1 for O-H bending. Broad peaks appeared at 3200-3600 cm-1 corresponding to OH and NH stretching  were evaluated for their flow property. From the values of angle of repose, it is evident that blends having graft copolymers showed better flow properties than that of blends having acetylated starch and native starch . Trial bStandard physical test of tablets2 and 0.53 to 0.84%, respectively. Tablets with all aforesaid composition passed USP criteria for friability (<1.00% w/w). Outcomes from friability assessment revealed excellent mechanical strength of the tablets. The weight variation of individual tablet was found to be within ± 5% w/w of average weight of tablets, which affirmed that all the tablets have passed the USP weight variation test.Tablets prepared (F1–F5) with mentioned array of excipients had thickness varying from 3.19-3.41 mm . Drug coIn-vitro drug release studiesin-vitro release profiles of salbutamol sulphate matrix tablets in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) are presented in The – group increased chain relaxation; and ionic nature of chains facilitated the water uptake resulting increase in the extent of swelling at higher pH. While using starch as a carrier, 80–85% of drug was released in SIF during the initial 1h and this trend decreased in case of acetylated starch to 41% of drug release. Further in case of St-g-PMMA and Ast-g-PMMA as a carrier, drug release reduced to 17.5% and 16.1% respectively. In SGF, a remarkable decrease was observed in graft copolymer matrices i.e. 9.5% in case of St-g-PMMA in initial 1h and decreased to 7.01% in case of Ast-g-PMMA. Comparison of drug release pattern from graft copolymers matrices (F3 and F4) with HPMC K100M containing tablets in SIF revealed that matrix tablets (F3 and F4) released 82.45% and 76.71% of drug respectively within 12 h while HPMC K100M tablets released 74.8% of drug content within 12 h. So, retardation of drug release rate of formulations having graft copolymers (F3 & F4) was almost equivalent to commercially used controlled release polymer i.e. HPMC K100M which supported their application as potential excipients in controlled drug delivery system.The drug release rate of graft copolymers was greater in SIF than in SGF perhaps due to enhanced swellability of carrier in SIF, because of ionization of carboxyl groups. The swelling is dependent on the intermolecular interaction between the polymer components of grafted copolymer. At lower pH, the carboxyl group of the grafted methyl methacrylate chain remains almost in unionized state might be due to involvement of hydrogen bond formation with hydroxyl group of starch, which made the polymer segments rigid and thereby retarded the water uptake and decreased the extent of swellability.Whereas at higher pH the carboxyl groups get ionized, which reduced the starch–acrylic interaction by decreasing H-bonding; the repulsion among similarly charged –COOin-vitro dissolution profiles of salbutamol sulphate from radiolabelled and unradiolabelled graft copolymer matrix tablets (F3 & F4) in the simulated intestinal fluid (pH 6.8) were investigated, in order to determine the possible effect of the labelling process on the kinetics of drug release from the matrix tablets. By analyzing the results compiled in 99mTc did not intervene with the drug release. In fact, it is distinctly mentioned that the dissolution profiles of labelled and unlabelled tablets were basically super imposable, suggesting that this technique did not affect the drug release rate. Actually, the cumulative amount of drug released from the tablets after 14 h was 90.64 ± 2.19% (unlabelled), and 87.11 ± 2.48% (labelled), for formulation F3; 80.67 ± 2.22% (unlabelled) and 77.96 ± 2.88% (labelled) for formulation F4.The Kinetics and mechanism of drug releasein-vitro dissolution data into various kinetic models and the values of release exponent (np), kinetic constant (K) and regression coefficient were determined, as indicated in The drug release mechanism was found by fitting the In-vitro release proﬁle of graft copolymers matrix formulations (F3 & F4) and HPMC K100M tablets (F5) were best expressed by the Higuchi model, as the plots showed high linearity with regression value of 0.995, 0.991, and 0.996 respectively. However, Higuchi’s equation failed to explain the influence of swelling of the matrix upon hydration and gradual erosion of the matrix. Therefore, the dissolution data were fitted into the well-known exponential Korsmeyer-peppas equation; here the value of release exponent (nP) explains the release mechanism of the drug from the matrix system. Observed ‘nP’ values for release profiles of formulation F3, F4 and F5 were in between 0.50 to 0.89 indicating anomalous release behavior coupled with diffusion and erosion. The release exponents for formulation F1 was found less than 0.5 indicating Quasi fickian diffusion mechanism for the drug release.Bio-analytical method validationSelectivityThe chromatograms of blank plasma and a spiked plasma sample are shown in Limit of detection (LOD) and Limit of quantification (LOQ)salbutamol sulphate were 33.97 ng/mL and 101.91 ng/mL, respectively. LOD and LOQ for LinearityStandard calibration curve in rabbit plasma was found to be linear at concentrations ranging from 100 to 1,200 ng/mL (Y = 0.0318x + 1.4027) with correlation coefficient of 0.9968.AccuracyRecovery studies were performed to validate the accuracy of developed method. To the preanalysed sample solution, a definite concentration of standard drug was added and then its recovery was analyzed. The method was found to be accurate if % recovery is 100 ± 15% and % CV is <15%. The % recovery of drug in plasma found in the range of 89.59 -92.41% and coefficient of variation was 2.21-3.81. Since the % recovery and % CV is within the range, it shows accuracy of method. PrecisionPrecision of the method was assessed by intra-day and inter-day analysis of six replicates for each concentration at 3 different concentration levels . The coefficient of variation (CVs) at each concentration level was expressed as precision. The method proved to be precise because the % CV for is not more than 5.61% at three different concentration levels. In-vivo pharmacokineticsmax) and corresponding peak time (tmax) were determined by assessing the individual serum drug concentration-time profiles. The elimination rate constants (Ke) were obtained from least square fitted terminal log-linear portion of the serum concentration-time profile. The elimination half life (t1/2) was calculated as 0.693/Ke. The area under the plasma concentration [AUC] 0–24 vs time curve was determined by linear trapezoidal rule until the last measurement point.The results of plasma drug concentration at different time intervals, after administration of tablets of formulation F1-F5 and Asthalin SA 8 mg tablets to rabbits, are shown in max of graft copolymer matrices F3 (578.9 ng/mL) and F4 (546.7 ng/mL) was significantly (p<0.05) lower than starch matrix F1 (924.1 ng/mL) and acetylated starch matrix tablets (812.4 ng/mL). Decrement in Cmax value of the controlled release tablets of graft copolymers F3 and F4 indicated their sustained and prolonged effect. The tmax of graft copolymer matrices F3 (6 h) and F4 (7.5 h) was significantly (p<0.05) higher than starch matrix F1 (1.5 h) and acetylated starch matrix tablets (2.5 h), which indicated slow absorption rate from the graft copolymer tablets due to extended release effect of hydrophobic polymer. Different elimination rate constants of various formulations may be due to difference in the metabolism pathway of polymer matrices. The elimination half life (t½) of the F3 (12.15 h) and F4 (13.07 h) was more than F1 (5.45 h) and F2 (6.72 h), which confirmed the prolonged availability of SS in graft copolymer matrix tablets. In all formulations, large difference was observed between plasma half life and tmax which reflects significant difference between absorption and elimination time of the drug.The Cmax, Cmax, AUC, Ke, and t1/2) of the graft copolymers matrices, HPMC K100M matrix tablets and commercial tablets. The results revealed that the graft copolymer matrices provided comparable sustained and prolonged effect as compared to HPMC K100M matrix tablets and marketed product . Therefore graft copolymers can be used as potential excipients in controlled drug delivery system.However there was no significant difference in the pharmacokinetic parameters , tablet starts dispersing corresponding to attenuate radioactivity  in rabbits in comparison with starch matrix tablets (F1). Scintigraphic images of starch matrix tablets (F1) clearly showed attenuation of the radioactivity within 1 h affected . This inactivity  and imagage form . The resin-vitro drug release rate was greater in SIF as compared to SGF which showed pH sensitivity and site-specific drug delivery of graft copolymer matrices. SIF facilitates ionization of the carboxyl groups of the grafted methyl methacrylate chain which enhances the repulsion between similarly charged COO– group rendering to chain relaxation; and ionic nature of chains facilitates water uptake, with the overall increase in the drug release in the dissolution medium of higher pH. In graft copolymers matrices, drug release was controlled by diffusion and erosion. The HPLC method used for in-vivo pharmacokinetic study has been validated according to U.S. Food and Drug Administration guidelines for bioanalytical methods and found to be linear, accurate and precise both in upper and lower concentration range with acceptable error and % CV values. In-vivo pharmacokinetic study revealed that in case of graft copolymer matrices (F3 & F4), value of tmax was higher and Cmax was lower than that of starch and acetylated starch matrix tablets (F1& F2). It reflects that graft copolymerization markedly influence the capacity of starch/acetylated starch in modulating drug release. Pharmacokinetic parameters of graft copolymers matrices (F3 & F4), HPMC K100M matrix tablets (F5) and marketed product were almost comparable, indicating ability of graft copolymers as promising vehicles for sustained release formulations in oral drug delivery system.From the present work, it can concluded that tablets prepared by salbutamol sulfate as a model drug and graft copolymer matrices as release retarding agent are capable of giving up drugs for a longer duration and their drug release characteristics are comparable with a controlled release matrix of HPMC K100M. Results of preformulation study revealed that graft copolymerization improved the flow property of native starch. Matrix tablets prepared by graft copolymers and HPMC K100M gave slow release of the drug up to14 h. The in-vitro and in-vivo studies were well correlated and confirm the ability for graft copolymer formulations to release the drug only after a desired period of time in a controlled manner.The Gamma scintigraphic images were showed the controlled release behavior of graft copolymer formulation which revealed their suitability for intestinal drug delivery system. Results of


Papaver somniferum L.), the only commercial source of morphinan alkaloids, has been used as a medicinal plant since ancient times. It produces benzylisoquinoline alkaloids (BIA) including the narcotic analgesic morphine, the muscle relaxant papaverine, and the anti-cancer agent noscapine. Though BIAs play crucial roles in many biological mechanisms their steps in biosynthesis and the responsible genes remain to be revealed. In this study, expressions of 3-hydroxy-N-methylcoclaurine 4′–methyltransferase (4′OMT) and reticuline 7-O-methyltransferase (7OMT) genes were subjected to manipulation to functionally characterize their roles in BIA biosynthesis. Measurements of alkaloid accumulation were performed in leaf, stem, and capsule tissues accordingly. Suppression of 4′OMT expression caused reduction in the total alkaloid content in stem tissue whereas total alkaloid content was significantly induced in the capsule. Silencing of the 7OMT gene also caused repression in total alkaloid content in the stem. On the other hand, over-expression of 4′OMT and 7OMT resulted in higher morphine accumulation in the stem but suppressed amount in the capsule. Moreover, differential expression in several BIA synthesis genes  were observed upon manipulation of 4′OMT and 7OMT expression. Upon silencing and overexpression applications, tissue specific effects of these genes were identified. Manipulation of 4′OMT and 7OMT genes caused differentiated accumulation of BIAs including morphine and noscapine in capsule and stem tissues.Alkaloids are diverse group of secondary metabolites generally found in plants. Opium poppy ( Papaver somniferum L.), belongs to the Papaveraceae family and has been used as a medicine or drug for a long time , commonly used for pharmaceutical purposes , converts (S)-reticuline to (S)-laudanine , codeinone reductase (COR), and codeine O-demethylase  and dopamine- catalyzed by tyrosine/DOPA decarboxylase (TYDC) to generate (S)-norcoclourine  were also quantified via qRT-PCR.In the current study, to address the tissue specific functional roles of Papaver somniferum cv Ofis 95, a morphine rich variety, seeds were provided from Toprak Mahsulleri Ofisi . These were potted in %25 peats, %25 perlite, and %50 soil mixtures, and then transferred to a growth chamber. Plants were kept with day/night cycles of 16/8 h at 20/24°C photoperiod and 3 month-old leaves were selected for gene cloning experiments.4′OMT2  and 7OMT (GenBank: FJ156103.1) complementary DNA sequences.Collected tissues were ground to a fine powder in liquid nitrogen. RNA isolations were performed by TRIzol® Reagent  following the manufacturer's instructions. Quantity of the isolated RNAs was measured using NanoDrop 2000c spectrophotometer  and the integrity of RNA was checked on 2% agarose gels. RNAs were reverse transcribed using Superscript III first strand cDNA synthesis kit  to specifically amplify OMT (GenBank: AY217334.1) (293 bp) and 7OMT (GenBank: FJ156103.1) (451 bp) were amplified with the primers listed in Supplementary Table BamhI and SmaI restriction enzyme to be used in the next step. The amplified 4′OMT and 7OMT genes were then inserted into pGEM-T vector . Both TRV and plasmid DNA were digested with BamHI and SmaI restriction endonucleases. The cleaved products were ligated into the TRV2  vectors. After cloning verification by sequencing, the constructs were separately transformed into Agrobacterium tumefaciens strain LBA4404 via electroporation. A. tumefaciens colonies separately transformed with TRV1 and pTRV2 with gene of interest were selected, then were grown in 10 mL of Luria-Bertani (LB) medium containing 50 mg/L kanamycin with overnight shaking at 250 rpm 28°C until cell absorbance reached to OD600 = 2. The cell cultures were centrifuged at 2000 g for 15 min, and the pellets were resuspended in 5 mL of induction buffer, , pH:5.8, until OD600 reached to 0.8 for TRV2 and 0.2 for TRV1. Agrobacterium LBA4404 strains consisting TRV1 and TRV2 were then mixed in a 1:1 ratio. The mixture was used for agro-infiltration assay by needleless syringe to young leaves of opium poppy. The tip of the syringe without a needle suppressed against the underside of a leaf while synchronously applying kindly counterpressure to the other side of the leaf. Later, Agrobacterium is injected into the air space along the inside of the leaf stomata. The control plants were also agro-infiltrated with TRV2-Empty vector. The inoculation repeated two times with 7 days interval. One week after the second inoculation, the samples were harvested and stored at −80°C until use. The number of infiltrated plants was 15 for each pTRV2-4′OMT and pTRV2-7OMT experiment and among three of them which were almost equally silenced and the empty vector selected for further analyzes.Virus Induced Gene Silencing (VIGS) was carried out using TRV-based vector system. Fragments of 4′4′OMT and 7OMT genes cloned into pGEM-T vector before cloned into a viral-based vector pGR106 containing 35S promoter. 4′OMT (1077 bp) and 7OMT (1170 bp) genes were amplified by PCR and ligated into pGEM-T vector. For this, forward primers containing NotI restriction site added the flag peptides (4′OMT-NotI FLAG and 7OMT-NotI FLAG) and reverse primers  were used for cloning  containing 1 mM MES, 150 mM acetosyringone, and 10 mM MgCl4′OMT and 7OMT in overexpressed and silenced tissues, qRT-PCR assays were performed using LightCycler 480 Real-Time PCR System . Additionally, the transcript levels of some BIA biosynthesis genes such as COR, SAT, TYDC, CNMT, 6-OMT were also measured. The primers used for measurements were given in Supplementary Table ΔCt−Δ method , respectively. The elution program was: 0–6 min, 40% B; 6–10 min, 50% B; 10–15 min, 90% B; 15–16 min, 90% B; 16–25 min, 40% B. All the measurements for each sample were triplicated.Plant materials were dried at 28°C for 2 days. Dried samples (0.1 g) were soaked in methanol  at room temperature for 1 day with shaking followed by filtration to separate the marc and evaporate. The extracts were solved in 2000 ppm and diluted to 1/200 ppm from stock. To analyze the HPLC-TOF/MS, the samples were filtered through 0.45 μm membranes. Morphine, codeine, thebaine, papaverine, noscapine, and laudanosine were analyzed using specific standards. Alkaloids were quantitated in Agilent 1260 Infinity HPLC system  coupled with Agilent 6210 TOF-MS detector and Agilent EC 250/4 Nucleosil 100–5 . The column temperature was adjusted at 35°C and the injection volume was set to 10 μL. Mobile phases A and B were water/1 mL L4′OMT and 7OMT genes in capsule, stem, and leaf tissues of opium poppy. Expression profiles of mRNAs, and alkaloids were analyzed accordingly using proper approaches. We observed correlated results between silenced and overexpressed tissues.In this study, we have successfully manipulated 4′OMT gene was obtained via agro-infiltration upon VIGS in opium poppy with the rates of 92, 71, and 46% in stem, capsule, and leaf tissue, respectively, .Effective silencing on , Figure . To measm Figure . The low, Figure . Silenci Figures , 6A.7OMT gene in BIA biosynthesis. 7OMT transcript was successfully suppressed in stem, capsule, and leaves with the rate of 30, 28, and 32%, respectively, . Among them, morphine content was remarkably suppressed approximately six-fold, whereas reduction of other alkaloids was about three-fold. In opposition, 7OMT VIGS in capsule caused induction of morphine levels. Meanwhile, no significant change was observed in total alkaloid accumulation. On the other hand, compared to morphine level, approximately 1.5-fold lower accumulation of thebaine and noscapine was measured in capsule. Upon 7OMT VIGS, it was observed that noscapine and codeine amounts were significantly reduced in leaf tissue .VIGS assay was also conducted to discover functional role of , Figure . Upon si, Figure . It was  Figures , 6A. Amo Figures , 6A.4′OMT gene was successfully overexpressed under the viral constitutive CaMV35S promoter. The highest overexpression level was found to be 475% in capsule . In stem, increased amount of (S)-reticuline was observed which caused 43% induction of total alkaloid content. Moreover, it was detected that both morphine and noscapine levels induced about two-fold. Total alkaloid measurement in leaf showed that any significant difference was present upon the overexpression. There was a minor alteration for noscapine content in overexpressed opium poppy. On the other hand, a considerable suppression of total alkaloid content (75%) was measured in capsule in response to 4′OMT overexpression. The highest reduction was detected for morphine and noscapine content .e Figure . Accordis Figure . The hig Figures , 6A. In  Figures , 6A.7OMT gene in all target tissues of opium poppy. Overexpression assay revealed induction of 7OMT in leaf, stem, and capsule with 497, 7614, and 471%, respectively, (Figure COR, SAT, 6OMT, and 4′OMT gene transcripts increased considerably in stem. However, CNMT, 6OMT, and COR levels were significantly decreased in capsule (Figure 6OMT and decrease of CNMT and TYDC expressions. Overexpression of 7OMT gene led to down-regulation of CNMT in all tissues (Figure 6OMT was observed between stem and capsule tissues. Its expression was induced in stem while a down-regulation was detected in capsule. The over-expression of 7OMT resulted in a considerable increase of alkaloid accumulation in stem and leaf tissues. Furthermore, higher morphine concentration was measured in stem compared to control plant. Additionally, overexpression of 7OMT reduced noscapine level remarkably in capsule (Figures Here, we performed overexpression of , Figure . The leve Figure . The ress Figure . A rever Figures , 6A.4′OMT silencing (Figure 7OMT overexpression caused increase of N, N-dimethylnarcotine in capsule.In addition to major BIAs, we also measured other types of alkaloids such as oripavine, N, N-dimethylnarcotine, dimethylpapaverine, and unidentified molecules in capsule and stem tissues Figure . N, N-dig Figure . On the Plants are being used as source of secondary metabolites for medicinal purposes. Opium poppy, an agronomically and medicinally important crop, produces opiates such as morphine and noscapine (Unver et al., 4′OMT and 7OMT were successfully manipulated in opium poppy. To date, while VIGS of 4′OMT and 7OMT was reported in opium poppy (Desgagné-Penix and Facchini, Here, to functionally analyze the regulatory roles in BIA biosynthesis in tissue specific manner, expression levels of CYP82Y1 gene in opium poppy resulted in diverse transcript levels in different opium poppy tissues (Dang and Facchini, Silencing and overexpression of genes involved in BIA biosynthesis led to the accumulation of an altered amount of alkaloids in opium poppy tissues Figures , 4C, 5C.4′OMT2 gene silencing caused reduction in all measured alkaloids in the stem and that total alkaloid content decreased by 41% (Figure 4′OMT2 in the stem resulted in a 43% total alkaloid reduction (Desgagné-Penix and Facchini, 4′OMT in the stem. Though, Inui et al. (4′OMT overexpression in Coptis japonica (Inui et al., C. japonica the overexpression of this gene had a lesser extent on total alkaloid accumulation. The inconsistency between our results and C. japonica overexpression outcomes might have resulted from the different metabolic branches of BIA pathway in host plants.% Figure . Similar% Figure  also repi et al.  measured4′OMT caused approximately a two-fold reduction whereas overexpression resulted in a two-fold induction of morphine content in stem (Figures 4′OMT leads consistent regulation on morphine content. On the other hand, the expression level differentiation of CNMT and SAT was found to be consistent with the manipulation of 4′OMT. Therefore, 4′OMT might positively regulate the expression of these genes by adjusting the morphine accumulation (Figures 4′OMT expression might be rate-limiting on BIA biosynthesis (Desgagné-Penix and Facchini, NMCH gene for BIA accumulation in opium poppy. It could be concluded that BIA biosynthesis rate-limiting regulation does not depend on only one gene (Frick et al., We detected correlating morphine content alteration upon gene silencing and overexpression. The silencing of  Figures , 4C. The Figures , 4. Like4′OMT gene. The accumulation of morphine, thebaine, and noscapine were increased in the capsule tissue upon 4′OMT VIGS. On the other hand, their production was suppressed by 4′OMT over expression. Though the expression level of COR and SAT genes were found to be down-regulated, in a capsule of 4′OMT they were silenced. It was previously stated that overexpression of COR and SAT genes caused total alkaloid accumulation in opium poppy (Allen et al., COR and SAT gene in the stem and then the transportation of intermediate products from stem to capsule tissue. Furthermore, it can be speculated that an unidentified pathway might have resulted in the accumulation of more alkaloids in the capsule.Different alkaloid profiles were analyzed in a capsule tissue from that of a leaf and stem by manipulation of 7OMT regulates laudanosine biosynthesis (Desgagné-Penix and Facchini, 7OMT has also considerable effect on biosynthesis of morphine, thebaine and noscapine. Due to different roles of stem and capsule tissues for BIA biosynthesis and accumulation, 7OMT gene might be involved in the biosynthesis of morphine at tissue specific manner. Manipulation of 7OMT altered the amount of morphine in stem. A similar pattern was observed for thebaine accumulation in stem by 7OMT manipulation (Figures 7OMT shows similar impact on biosynthesis of these alkaloids. Surprisingly, we showed the regulatory action of 7OMT on noscapine biosynthesis, which is placed on a different branch then morphinan alkaloids. Moreover, opposite levels of noscapine were measured between stem and capsule upon manipulation of 7OMT implying tissue specific regulation of 7OMT. Manipulation of 7OMT has impact on expression of 6OMT at different levels in tissues (Figures 7OMT VIGS we observed the suppression of 6OMT expression and reduction in total alkaloid amount (Figures 6OMT VIGS (Desgagné-Penix and Facchini, 7OMT-silenced capsule tissue supporting tissue specific action of 7OMT. Since the whole BIA pathway is still unclear, these outcomes might be sourced by putative unknown pathways. As discussed by Leonard et al. (Up to date, it has been reported that silencing of  Figures , 5C. Our Figures , 5B. In  Figures . Similar Figures  found ded et al. , metabold et al. .COR transcript level and thebaine accumulation in stem for both 4′OMT and 7OMT assays suggests that the COR encoding gene may have an important effect on the biosynthesis of thebaine. The results showed that where the COR down-regulated thebaine content was decreased or the up-regulation of COR induced the thebaine levels in stem and capsule with the exception 7OMT induced capsule. Consistent with our results, the silencing of COR caused a decrease in salutaridine, thebaine, codeine, morphine in latex (Wijekoon and Facchini, COR and SAT genes altered the alkaloid levels of intermediates (Allen et al., SAT transcript leads to the accumulation of morphinan alkaloids such as morphine, thebaine, and codeine. SAT transcripts levels in stem revealed a correlation especially with morphine accumulation for both suppression and over-expression assays but the same similarity could not be found for other alkaloids. Therefore, both 4′OMT and 7OMT might have an effect on SAT transcript in biosynthesis of morphine.An unexpected correlation between 4′OMT and 7OMT in BIA biosynthesis. Upon silencing and overexpression applications, tissue specific effects of these genes were identified. Manipulation of 4′OMT and 7OMT genes caused differentiated accumulation of BIAs including morphine and noscapine in capsule and stem tissues.In the presented study, through the metabolic engineering approaches, we present new results about the regulatory roles for TG, MT, and TU drafted the paper, MT and TU analyzed the data, TU, IP, and SÖ organized and materials and planned the study. EO and TU performed experiments. ID conducted the metabolite measurements. Aİ and SO helped plant growth and inoculation assays.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.