Measurement of multi-segment foot joint angles during gait using a wearable system.

Usually the measurement of multi-segment foot and ankle complex kinematics is done with stationary motion capture devices which are limited to use in a gait laboratory. This study aimed to propose and validate a wearable system to measure the foot and ankle complex joint angles during gait in daily conditions, and then to investigate its suitability for clinical evaluations. The foot and ankle complex consisted of four segments (shank, hindfoot, forefoot, and toes), with an inertial measurement unit (3D gyroscopes and 3D accelerometers) attached to each segment. The angles between the four segments were calculated in the sagittal, coronal, and transverse planes using a new algorithm combining strap-down integration and detection of low-acceleration instants. To validate the joint angles measured by the wearable system, three subjects walked on a treadmill for five minutes at three different speeds. A camera-based stationary system that used a cluster of markers on each segment was used as a reference. To test the suitability of the system for clinical evaluation, the joint angle ranges were compared between a group of 10 healthy subjects and a group of 12 patients with ankle osteoarthritis, during two 50-m walking trials where the wearable system was attached to each subject. On average, over all joints and walking speeds, the RMS differences and correlation coefficients between the angular curves obtained using the wearable system and the stationary system were 1 deg and 0.93, respectively. Moreover, this system was able to detect significant alteration of foot and ankle function between the group of patients with ankle osteoarthritis and the group of healthy subjects. In conclusion, this wearable system was accurate and suitable for clinical evaluation when used to measure the multi-segment foot and ankle complex kinematics during long-distance walks in daily life conditions.

CHO cell engineering to prevent polypeptide aggregation and improve therapeutic protein secretion.

The ability to efficiently produce recombinant proteins in a secreted form is highly desirable and cultured mammalian cells such as CHO cells have become the preferred host as they secrete proteins with human-like post-translational modifications. However, attempts to express high levels of particular proteins in CHO cells may consistently result in low yields, even for non-engineered proteins such as immunoglobulins. In this study, we identified the responsible faulty step at the stage of translational arrest, translocation and early processing for such a "difficult-to-express" immunoglobulin, resulting in improper cleavage of the light chain and its precipitation in an insoluble cellular fraction unable to contribute to immunoglobulin assembly. We further show that proper processing and secretion were restored by over-expressing human signal receptor protein SRP14 and other components of the secretion pathway. This allowed the expression of the difficult-to-express protein to high yields, and it also increased the production of an easy-to-express protein. Our results demonstrate that components of the secretory and processing pathways can be limiting, and that engineering of the secretory pathway may be used to improve the secretion efficiency of therapeutic proteins from CHO cells.

In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications.

We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.

Segmentation of foot and ankle complex based on kinematic criteria.

Although various foot models were proposed for kinematics assessment using skin makers, no objective justification exists for the foot segmentations. This study proposed objective kinematic criteria to define which foot joints are relevant (dominant) in skin markers assessments. Among the studied joints, shank-hindfoot, hindfoot-midfoot and medial-lateral forefoot joints were found to have larger mobility than flexibility of their neighbour bonesets. The amplitude and pattern consistency of these joint angles confirmed their dominancy. Nevertheless, the consistency of the medial-lateral forefoot joint amplitude was lower. These three joints also showed acceptable sensibility to experimental errors which supported their dominancy. This study concluded that to be reliable for assessments using skin markers, the foot and ankle complex could be divided into shank, hindfoot, medial forefoot, lateral forefoot and toes. Kinematics of foot models with more segments must be more cautiously used.

Muscle moment-arms: a key element in muscle-force estimation.

A clear and rigorous definition of muscle moment-arms in the context of musculoskeletal systems modelling is presented, using classical mechanics and screw theory. The definition provides an alternative to the tendon excursion method, which can lead to incorrect moment-arms if used inappropriately due to its dependency on the choice of joint coordinates. The definition of moment-arms, and the presented construction method, apply to musculoskeletal models in which the bones are modelled as rigid bodies, the joints are modelled as ideal mechanical joints and the muscles are modelled as massless, frictionless cables wrapping over the bony protrusions, approximated using geometric surfaces. In this context, the definition is independent of any coordinate choice. It is then used to solve a muscle-force estimation problem for a simple 2D conceptual model and compared with an incorrect application of the tendon excursion method. The relative errors between the two solutions vary between 0% and 100%.

Human fetal bone cells in delivery systems for bone engineering.

The aim of this study was to culture human fetal bone cells (dedicated cell banks of fetal bone derived from 14 week gestation femurs) within both hyaluronic acid gel and collagen foam, to compare the biocompatibility of both matrices as potential delivery systems for bone engineering and particularly for oral application. Fetal bone cell banks were prepared from one organ donation and cells were cultured for up to 4 weeks within hyaluronic acid (Mesolis(®)) and collagen foams (TissueFleece(®)). Cell survival and differentiation were assessed by cell proliferation assays and histology of frozen sections stained with Giemsa, von Kossa and ALP at 1, 2 and 4 weeks of culture. Within both materials, fetal bone cells could proliferate in three-dimensional structure at ∼70% capacity compared to monolayer culture. In addition, these cells were positive for ALP and von Kossa staining, indicating cellular differentiation and matrix production. Collagen foam provides a better structure for fetal bone cell delivery if cavity filling is necessary and hydrogels would permit an injectable technique for difficult to treat areas. In all, there was high biocompatibility, cellular differentiation and matrix deposition seen in both matrices by fetal bone cells, allowing for easy cell delivery for bone stimulation in vivo. Copyright © 2011 John Wiley & Sons, Ltd.

Covalent cell surface functionalization of human fetal osteoblasts for tissue engineering.

The chemical functionalization of cell-surface proteins of human primary fetal bone cells with hydrophilic bioorthogonal intermediates was investigated. Toward this goal, chemical pathways were developed for click reaction-mediated coupling of alkyne derivatives with cellular azido-expressing proteins. The incorporation via a tetraethylene glycol linker of a dipeptide and a reporter biotin allowed the proof of concept for the introduction of cell-specific peptide ligands and to follow the reaction in living cells. Tuning the conditions of the click reaction resulted in chemical functionalization of living human fetal osteoblasts with excellent cell survival.

Gene transfer engineering for astrocyte-specific silencing in the CNS.

Cell-type-specific gene silencing is critical to understand cell functions in normal and pathological conditions, in particular in the brain where strong cellular heterogeneity exists. Molecular engineering of lentiviral vectors has been widely used to express genes of interest specifically in neurons or astrocytes. However, we show that these strategies are not suitable for astrocyte-specific gene silencing due to the processing of small hairpin RNA (shRNA) in a cell. Here we develop an indirect method based on a tetracycline-regulated system to fully restrict shRNA expression to astrocytes. The combination of Mokola-G envelope pseudotyping, glutamine synthetase promoter and two distinct microRNA target sequences provides a powerful tool for efficient and cell-type-specific gene silencing in the central nervous system. We anticipate our vector will be a potent and versatile system to improve the targeting of cell populations for fundamental as well as therapeutic applications.