Pre-clinical validation of a new partially cemented femoral prosthesis by synergetic use of numerical and experimental methods

The present work reports the pre-clinical validation of an innovative partially cemented femoral prosthesis called cement-locked uncemented (CLU) prosthesis. The inventors of the device under investigation claimed that, when compared to a comparable fully cemented stem, the new stem would present various advantages. Two previous experimental studies confirmed that primary stability and stress shielding were comparable to those of cemented stems. Aim of the present study was to investigate if the remaining claims were confirmed as well. A complete finite element model of the bone-implant complex was created from CT data. The model was validated against in vitro measurements of bone surface strains as well as against primary stability measurements. The peak stresses predicted in the CLU cement mantle were not found significantly lower than those reported in other studies on fully cemented stems. However, once the cement inlet geometry is optimised and the associated stress risers are eliminated, the CLU cement mantle should be subjected to much lower stresses. The stress induced in the stems by both load cases was well below the fatigue limit of the Ti6Al4V alloy. Finite element models predicted for all load cases relative motion between cement and metal lower than 60 microm. This amplitude may be fully accommodated by elastic deformations of the cement micro-ridges. The experimental and numerical results showed the validity of the new fixation concept, although a further optimisation of the geometry of the cement pockets is needed in order to further reduce the stresses in the cement.     

Proximal femoral surface strain gauge analysis of a new epiphyseal prosthesis

This paper describes a new type of proximal femoral, replacement arthroplasty and its preliminary, pre-clinical, evaluation. The prosthesis was designed as a replacement for the femoral capital epiphysis. It comprises a thin, metal, articular shell and an underlying ‘epiphyseal’ replacement which is modulus-matched to adjacent proximal femoral bone. Surface strain gauge analysis of the proximal femur under conditions simulating static single leg stance demonstrated that the new prosthesis maintains an essentially normal femoral strain distribution. It is hoped that selective load transmissions across the prosthesis-bone interface should result in less post-implantation proximal bone resorption.     

Analysis of a femoral hip prosthesis designed to reduce stress shielding

The natural stress distribution in the femur is significantly altered after total hip arthroplasty (THA). When an implant is introduced, it will carry a portion of the load, causing a reduction of stress in some regions of the remaining bone. This phenomenon is commonly known as stress shielding. In response to the changed mechanical environment the shielded bone will remodel according to Wolff's law, resulting in a loss of bone mass through the biological process called resorption. Resorption can, in turn, cause or contribute to loosening of the prosthesis. The problem is particularly common among younger THA recipients. This study explores the hypothesis that through redesign, a total hip prosthesis can be developed to substantially reduce stress shielding. First, we describe the development of a new femoral hip prosthesis designed to alleviate this problem through a new geometry and system of proximal fixation. A numerical comparison with a conventional intramedullary prosthesis as well as another proximally fixed prosthesis, recently developed by Munting and Verhelpen (1995. Journal of Biomechanics 28(8), 949–961) is presented. The results show that the new design produces a more physiological stress state in the proximal femur.     

Vertical stiffness during one-legged hopping with and without using a running-specific prosthesis

Although athletes with unilateral below-the-knee amputations (BKAs) generally use their affected leg, including their prosthesis, as their take-off leg for the long jump, little is known about the spring-like leg behavior and stiffness regulation of the affected leg. The purpose of this study was to investigate vertical stiffness during one-legged hopping in an elite-level long jump athlete with a unilateral BKA. We used the spring-mass model to calculate vertical stiffness, which equals the ratio of maximum vertical ground reaction force to maximum center of mass displacement, while the athlete with a BKA hopped on one leg at a range of frequencies. Then, we compared the vertical stiffness of this athlete to seven non-amputee elite-level long-jumpers. We found that from 1.8 to 3.4 Hz, the vertical stiffness of the unaffected leg for an athlete with a BKA increases with faster hopping frequencies, but the vertical stiffness of the affected leg remains nearly constant across frequencies. The athlete with a BKA attained the desired hopping frequencies at 2.2 and 2.6 Hz, but was unable to match the lowest (1.8 Hz) and two highest frequencies (3.0 and 3.4 Hz) using his affected leg. We also found that at 2.5 Hz, unaffected leg vertical stiffness was 15% greater than affected leg vertical stiffness, and the vertical stiffness of non-amputee long-jumpers was 32% greater than the affected leg vertical stiffness of an athlete with a BKA. The results of the present study suggest that the vertical stiffness regulation strategy of an athlete with a unilateral BKA is not the same in the unaffected versus affected legs, and compared to non-amputees.     

Speed adaptation in a powered transtibial prosthesis controlled with a neuromuscular model

Control schemes for powered ankle-foot prostheses would benefit greatly from a means to make them inherently adaptive to different walking speeds. Towards this goal, one may attempt to emulate the intact human ankle, as it is capable of seamless adaptation. Human locomotion is governed by the interplay among legged dynamics, morphology and neural control including spinal reflexes. It has been suggested that reflexes contribute to the changes in ankle joint dynamics that correspond to walking at different speeds. Here, we use a data-driven muscle-tendon model that produces estimates of the activation, force, length and velocity of the major muscles spanning the ankle to derive local feedback loops that may be critical in the control of those muscles during walking. This purely reflexive approach ignores sources of non-reflexive neural drive and does not necessarily reflect the biological control scheme, yet can still closely reproduce the muscle dynamics estimated from biological data. The resulting neuromuscular model was applied to control a powered ankle-foot prosthesis and tested by an amputee walking at three speeds. The controller produced speed-adaptive behaviour; net ankle work increased with walking speed, highlighting the benefits of applying neuromuscular principles in the control of adaptive prosthetic limbs.     

Design and validation of a machine for reproducible precision insertion of femoral hip prostheses for preclinical testing

Preclinical testing of orthopaedic implants is becoming increasingly important to eliminate inferior designs before animal experiments or clinical trials are begun. Preclinical tests can include both laboratory bench tests and computational modeling. One problem with bench tests is that variability in prosthesis insertion can significantly influence the failure rate; this makes comparison of prostheses more difficult. To solve this problem an insertion method is required that is both accurate and reproducible. In this work, a general approach to the insertion of hip prostheses into femoral bones is proposed based on physically replicating an insertion path determined using computer animation. As a first step, the seated prosthesis position is determined from templates and femur radiographs. Three-dimensional images of the prosthesis and bone are then imported into computer animation software and an insertion path in the coronal plane is determined. The insertion path is used to determine the profile of a cam. By attaching the prosthesis to a carriage, which is pneumatically moved along this cam, the required insertion motion of the prosthesis in the coronal plane can be achieved. This paper describes the design and validation of the insertion machine. For the validation study, a nonsymmetric hip prosthesis design (Lubinus SPII, Waldemar Link, Germany) is used. It is shown that the insertion machine has sufficient accuracy and reproducibility for preclinical mechanical testing.     

The effect of three-dimensional shape optimization on the probabilistic response of a cemented femoral hip prosthesis

Probabilistic analyses allow the effect of uncertainty in system parameters on predicted model performance measures to be determined. Furthermore, using performance functions to describe a failure event, the probability of failure can be quantified. The effect of three-dimensional prosthesis shape optimization on the probabilistic response and failure probability of a cemented hip prosthesis system is investigated. Random variables include joint and muscle loading, cortical and cancellous bone and PMMA bone cement elastic properties, and strength parameters describing failure of the bone cement and the prosthesis-bone cement interface. Several performance functions describing the bone cement and prosthesis-cement interface are used to compute the probability of failure. When evaluated deterministically, most performance functions indicated a safe design, with the exception of interface tensile failure. However, when evaluated probabilistically, finite probabilities of failure were computed, some significant. The most likely mode of failure before shape optimization was prosthesis-bone cement interface tensile failure with a predicted probability of failure of 97.9%. Deterministic prosthesis shape optimization reduced the probability of failure for all performance functions and reduced prosthesis-bone cement interface tensile failure by 31.7%. Probability sensitivity factors indicate that the uncertainty in the joint loading, cement strength, and implant-cement interface strength have the greatest effect on the computed probability of failure. Implant shape optimization results in a more robust implant design that is less sensitive to uncertainties in joint loading, which cannot be easily controlled, and more sensitive to cement and interface properties, which are easier to modify.     

Development and validation of a HPLC method for the quantification of baculovirus particles

A HPLC method using an anion exchange column was developed for the quantification of baculovirus particles. To properly detect the virus eluting from the column, a nucleic acid dye was used to amplify the signal projected by the virus. The viral genome was labeled by incubating the virus with SYBR Green I at 37°C for a minimum of 1h. The virus was specifically eluted from the contaminants in 8.9 min at a NaCl concentration of 480 mM NaCl (in 20 mM Tris-HCl, pH 7.5). The total run time of the method was 25 min. The method resulted in a linear response from 1×10(8) to 5.0×10(10)viral particles (VP/ml). The detection limit was 3.0×10(7) and the quantification limit was 1×10(8)VP/ml. The intra-assay precision was <10% for both purified and crude virus preparations whereas the inter-assay precisions were <5% and <10% for purified and crude virus preparations, respectively. The recovery/accuracy of the method ranged from 78 to 101%. This method is a robust monitoring tool to facilitate research activities with baculovirus vector and accelerate development of baculovirus-based processes for manufacturing of biologics.     

Measuring multi-joint stiffness during single movements: numerical validation of a novel time-frequency approach

This study presents and validates a Time-Frequency technique for measuring 2-dimensional multijoint arm stiffness throughout a single planar movement as well as during static posture. It is proposed as an alternative to current regressive methods which require numerous repetitions to obtain average stiffness on a small segment of the hand trajectory. The method is based on the analysis of the reassigned spectrogram of the arm's response to impulsive perturbations and can estimate arm stiffness on a trial-by-trial basis. Analytic and empirical methods are first derived and tested through modal analysis on synthetic data. The technique's accuracy and robustness are assessed by modeling the estimation of stiffness time profiles changing at different rates and affected by different noise levels. Our method obtains results comparable with two well-known regressive techniques. We also test how the technique can identify the viscoelastic component of non-linear and higher than second order systems with a non-parametrical approach. The technique proposed here is very impervious to noise and can be used easily for both postural and movement tasks. Estimations of stiffness profiles are possible with only one perturbation, making our method a useful tool for estimating limb stiffness during motor learning and adaptation tasks, and for understanding the modulation of stiffness in individuals with neurodegenerative diseases.     

Qualification of a novel inline spiking method for virus filter validation

Virus filters are widely used in bioprocessing to reduce the risk of virus contamination in therapeutics. The small pores required to retain viruses are sensitive to plugging by trace contaminants and frequently require inline adsorptive prefiltration. Virus spiking studies are required to demonstrate virus removal capabilities of the virus filter using scale down filters. If prefiltration removes viruses and interferes with the measurement of virus filter LRV, the standard approach is to batch prefilter the protein solution, spike with virus, and then virus filter. For a number of proteins, batch prefiltration leads to increased plugging and significantly lower throughputs than inline prefiltration. A novel inline spiking method was developed to overcome this problem. This method allows the use of inline prefiltration with direct measurement of virus filter removal capabilities. The equipment and its operation are described. The method was tested with three different protein feeds, two different parvovirus filters, two virus injection rates; a salt spike, a bacteriophage spike, and two mammalian virus spikes: MMV and xMuLV. The novel inline method can reliably measure LRV at throughputs representative of the manufacturing process. It is recommended for applications where prefiltration is needed to improve throughput, prefiltration significantly reduces virus titer, and virus filter throughput is significantly reduced using batch vs. inline prefiltration. It can even help for the case where the virus preparation causes premature plugging.     

The validation of a bioanalytical method for the determination of clopidogrel in human plasma

A fast, sensitive and specific LC-MS/MS bioanalytical method for the determination of unchanged clopidogrel in human plasma has been developed and validated over the range of 10-12,000 pg mL(-1) (r2 0.9993) by the Contract Research group at HFL. Samples (0.3 mL) were buffered (pH 6.8), extracted using diethyl ether and 10 microL of the sample extract was injected onto the LC-MS/MS system. Analysis was performed using a C8 column (temperature controlled to 50 degrees C) by gradient elution at a flow rate of 0.9 mL min(-1) over a 3 min run time. Retention times of 1.61 and 1.59 min were observed for clopidogrel and 2H3-clopidogrel (I.S.), respectively. Detection was achieved using a Sciex API 4000, triple quadrupole mass spectrometer, in positive TurboIonspray (electrospray) ionisation mode. Ion transitions were monitored using MRM (multiple reaction monitoring) for clopidogrel (m/z 322-212) and for 2H3-clopidogrel (m/z 327-217). This validated method was used to support a pharmacokinetic study in healthy volunteers.     

A simple method for measuring stiffness during running

The spring-mass model, representing a runner as a point mass supported by a single linear leg spring, has been a widely used concept in studies on running and bouncing mechanics. However, the measurement of leg and vertical stiffness has previously required force platforms and high-speed kinematic measurement systems that are costly and difficult to handle in field conditions. We propose a new "sine-wave" method for measuring stiffness during running. Based on the modeling of the force-time curve by a sine function,this method allows leg and vertical stiffness to be estimated from just a few simple mechanical parameters: body mass, forward velocity, leg length, flight time, and contact time. We compared this method to force-platform-derived stiffness measurements for treadmill dynamometer and overground running conditions, at velocities ranging from 3.33 m.s-1 to maximal running velocity in both recreational and highly trained runners. Stiffness values calculated with the proposed method ranged from 0.67 % to 6.93 % less than the force platform method, and thus were judged to be acceptable. Furthermore, significant linear regressions (p < 0.01) close to the identity line were obtained between force platform and sine-wave model values of stiffness. Given the limits inherent in the use of the spring-mass model, it was concluded that this sine-wave method allows leg and stiffness estimates in running on the basis of a few mechanical parameters, and could be useful in further field measurements.     

Development and validation of a simple method for the extraction of human skin melanocytes

Primary melanocytes in culture are useful models for studying epidermal pigmentation and efficacy of melanogenic compounds, or developing advanced therapy medicinal products. Cell extraction is an inevitable and critical step in the establishment of cell cultures. Many enzymatic methods for extracting and growing cells derived from human skin, such as melanocytes, are described in literature. They are usually based on two enzymatic steps, Trypsin in combination with Dispase, in order to separate dermis from epidermis and subsequently to provide a suspension of epidermal cells. The objective of this work was to develop and validate an extraction method of human skin melanocytes being simple, effective and applicable to smaller skin samples, and avoiding animal reagents. TrypLE? product was tested on very limited size of human skin, equivalent of multiple 3-mm punch biopsies, and was compared to Trypsin/Dispase enzymes. Functionality of extracted cells was evaluated by analysis of viability, morphology and melanin production. In comparison with Trypsin/Dispase incubation method, the main advantages of TrypLE? incubation method were the easier of separation between dermis and epidermis and the higher population of melanocytes after extraction. Both protocols preserved morphological and biological characteristics of melanocytes. The minimum size of skin sample that allowed the extraction of functional cells was 6 × 3-mm punch biopsies (e.g., 42 mm²) whatever the method used. In conclusion, this new procedure based on TrypLE? incubation would be suitable for establishment of optimal primary melanocytes cultures for clinical applications and research.     

Towards a fibrous composite with dynamically controlled stiffness: lessons from echinoderms

Sea urchins and sea cucumbers, like other echinoderms, control the tensile properties of their connective tissues by regulating stress transfer between collagen fibrils. The collagen fibrils are spindle-shaped and up to 1 mm long with a constant aspect ratio of approx. 2000. They are organized into a tissue by an elastomeric network of fibrillin microfibrils. Interactions between the fibrils are regulated by soluble macromolecules that are secreted by local, neurally controlled, effector cells. We are characterizing the non-linear viscoelastic properties of sea cucumber dermis under different conditions, as well as the structures, molecules and molecular interactions that determine its properties. In addition, we are developing reagents that will bind covalently to fibril surfaces and reversibly form cross-links with other reagents, resulting in a chemically controlled stress-transfer capacity. The information being developed will lead to the design and construction of a synthetic analogue composed of fibres in an elastomeric matrix that contains photo- or electro-sensitive reagents that reversibly form interfibrillar cross-links.     

Protein structure validation using a semi-empirical method

Current practice of validating predicted protein structural model is knowledge-based where scoring parameters are derived from already known structures to obtain decision on validation out of this structure information. For example, the scoring parameter, Ramachandran Score gives percentage conformity with steric-property higher value of which implies higher acceptability. On the other hand, Force-Field Energy Score gives conformity with energy-wise stability higher value of which implies lower acceptability. Naturally, setting these two scoring parameters as target objectives sometimes yields a set of multiple models for the same protein for which acceptance based on a particular parameter, say, Ramachandran score, may not satisfy well with the acceptance of the same model based on other parameter, say, energy score. The confusion set of such models can further be resolved by introducing some parameters value of which are easily obtainable through experiment on the same protein. In this piece of work it was found that the confusion regarding final acceptance of a model out of multiple models of the same protein can be removed using a parameter Surface Rough Index which can be obtained through semi-empirical method from the ordinary microscopic image of heat denatured protein.     

RNAi in Haemonchus contortus: a potential method for target validation

RNA interference (RNAi) is a method for the functional analysis of specific genes, and is particularly well developed in the free-living nematode Caenorhabditis elegans. There have been several attempts to apply this method to parasitic nematodes. In a recent study undertaken in Haemonchus contortus, Geldhof and colleagues concluded that, although a mechanism for RNAi existed, the methods developed for RNAi in C. elegans had variable efficacy in this parasitic nematode. The potential benefits of RNAi are clear; however, further studies are required to characterize the mechanism present in parasitic nematodes, and to improve culture systems for these nematodes to monitor the long-term effects of RNAi. Only then could RNAi become a reliable assay of gene function.     

A method for the identification of in-vivo segmental stiffness properties of the spine

A numerical algorithm is used to estimate in-vivo segmental stiffness properties of individual spine segments based upon existing load-displacement data. A static nonlinear finite element model stimulates a pathological spine and corrective instrumentation system. A systematic procedure for establishing the model's stiffness parameters is described, in the form of a nonlinear constrained optimal design problem. The numerical method is demonstrated using as an example a case of adolescent idiopathic scoliosis requiring corrective surgery.     

Precision measurements of the RSA method using a phantom model of hip prosthesis

Radiostereometric analysis (RSA) has become one of the recommended techniques for pre-market evaluation of new joint implant designs. In this study we evaluated the effect of repositioning of X-ray tubes and phantom model on the precision of the RSA method. In precision measurements, we utilized mean error of rigid body fitting (ME) values as an internal control for examinations. ME value characterizes relative motion among the markers within each rigid body and is conventionally used to detect loosening of a bone marker. Three experiments, each consisting of 10 double examinations, were performed. In the first experiment, the X-ray tubes and the phantom model were not repositioned between one double examination. In experiments two and three, the X-ray tubes were repositioned between one double examination. In addition, the position of the phantom model was changed in experiment three. Results showed that significant differences could be found in 2 of 12 comparisons when evaluating the translation and rotation of the prosthetic components. Repositioning procedures increased ME values mimicking deformation of rigid body segments. Thus, ME value seemed to be a more sensitive parameter than migration values in this study design. These results confirmed the importance of standardized radiographic technique and accurate patient positioning for RSA measurements. Standardization and calibration procedures should be performed with phantom models in order to avoid unnecessary radiation dose of the patients. The present model gives the means to establish and to follow the intra-laboratory precision of the RSA method. The model is easily applicable in any research unit and allows the comparison of the precision values in different laboratories of multi-center trials.