Modulation of muscle contraction by a cell-permeable peptide

In contrast to immortal cell lines, primary cells are hardly susceptible to intracellular delivery methods such as transfection. In this study, we evaluated the direct delivery of several cell-permeable peptides under noninvasive conditions into living primary adult rat cardiomyocytes. We specifically monitored the functional effects of a cell-permeable peptide containing the 15 amino acid N-terminal peptide from human ventricular light chain-1 (VLC-1) on contraction and intracellular Ca2+ signals after electrical stimulation in primary adult cardiomyocytes. The transducible VLC-1 variant was taken up by cardiomyocytes within 5 min with more than 95% efficiency and localized to sarcomeric structures. Analysis of the functional effects of the cell-permeable VLC-1 revealed an enhancement of the intrinsic contractility of cardiomyocytes without affecting the intracellular Ca2+. Therefore, peptide transduction mediated by cell-penetrating peptides represents not only a unique strategy to enhance heart muscle function with no secondary effect on intracellular Ca2+ but also an invaluable tool for the modulation and manipulation of protein interactions in general and in primary cells.
Introduction
Therapeutical delivery of protein or peptides into living primary cells is hampered by the lack of a suitable and efficient method for the introduction of macromolecules. Most commonly used transfections methods work in immortalized cells but mostly fail in primary cells or require specialized and time-consuming protocols, i.e., production of viral vectors. Moreover, the limited life span of primary cells often does not provide sufficient time for expression of gene products and subsequent analysis of their effects. Physical methods such as microinjection and bead loading are highly invasive procedures and are not tolerated by several cell types. It has been known for quite some time that basic proteins like histones or poly-ornithine as well as stretches of basic amino acids within proteins aid the uptake of proteins into mammalian cells [14, 17, 20, 30]. In the 1990s, transducible peptides like penetratin-1 from the homeodomain of Antennapedia [11] and amino acids 48–57 from the transactivator of transcription of human immunodeficiency virus (HIV)-1 [14] were identified and exploited to introduce drugs or biological macromolecules into mammalian cells. Together with other native and synthetic transducing peptides, they are referred to as cell-penetrating peptides (CPPs) [12]. However, it became evident that the invasive methods used to analyze the transduction mediated by CPPs or effects of interconnected cargoes had led to overestimated transduction results and overrated effects of cargoes fused to CPPs [27]. Nevertheless, CPPs mediate the introduction of fused cargoes into living cells, with cargo-dependent mechanistical differences. One uptake mechanism can be described as a slow adsorptive endocytosis and is preferentially used by globular proteins, whereas small compounds like peptides linked to CPPs favor a rapid membrane potential-dependent uptake with overall intracellular availability of the transduced species [33]. CPP-mediated peptide transduction has recently been used to deliver cardioprotective peptides derived from protein kinase C [4, 7–9, 24], the antiapoptotic BH4 peptide derived from the apoptosis regulator protein Bcl-xL [26, 28] and a Nox2-derived peptide [34] into isolated cardiomyocytes or whole hearts. We have tested the feasibility of this approach to study muscle function in living adult primary cardiomyocytes. Therefore, we targeted the interaction between actin and the essential myosin light chain (MLC-1) of cardiac type II myosin by a peptide competition approach. Transgenic overexpression of the N terminus of the human ventricular MLC-1 (residues 1–15; hereafter termed VLC-1), which binds to actin and targets actin/MLC-1 interaction, significantly increased the magnitude and kinetics of the contraction of isolated perfused hearts [21]. We fused the same human VLC-1 peptide to the TAT CPP (VLC-1-TAT) and investigated its uptake, intracellular distribution, and functional consequences in primary living adult rat cardiomyocytes.
Materials and methods
Isolation of adult rat cardiomyocytes Primary cultures of cardiomyocytes from male WKY rats aged 3 months were performed as described previously [1].
Peptides The peptides consisted of either l-amino acids (capital letters) or of d-amino acids (lower case letters). In case of d-amino acid peptides, functional motifs were synthesized as retro-all d-variants (underlined) to maintain their functionality. Peptides were either labeled with the fluorophore 5,6-carboxy-tetramethylrhodamine (TAMRA) or fluorescein isothiocyanate (FITC). TAT (TAMRA- or FITC-rrrqrrkkrg), PTD4 [23, 33] (TAMRA-araqraaaray), and TAT-p21 (TAMRA-rrrqrrkkrgaaAGRKRRQTSMTDFYHSKRRLIFSa-amide) [33] were labeled directly at their N termini with the indicated fluorophores (Peptide Speciality Laboratories, Heidelberg). In the case of VLC-1 (MAPKKPEPKKSSAKA-C-TAMRA-ME-amide) and VLC-1-TAT (MAPKKPEPKDDAKAPAGRKKRRQRRR-C-TAMRA-ME-amide; Biosyntan, Berlin) the TAMRA fluorophore was coupled via an additional cysteine at the C terminus leaving the N terminus free for the interaction with actin.
Uptake of transducible peptides and controls To analyze the transduction ability of different compounds into living adult ventricular cardiomyocytes, the primary cells were plated into a laminin-coated μ-slide eight-well ibiTreat (ibidi, Martinsried) or four-well labtek (Nunc, Wiesbaden) microscopy observation chamber. The respective peptides and controls were added directly to Hank’s balanced salts solution buffered with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid at pH 7.4 (HBSS) or medium and gently shaken to yield a final concentration of 10 μM in case of the peptides and 5,6-TAMRA-fluorophore. Trypan blue (Sigma-Aldrich) was added to a final concentration of 0.5% (v/v) to the HBSS buffer. For the experiments summarized in Fig. 1 confocal images were acquired 1 h after addition of the respective compounds to living cardiomyocytes. To study the intracellular localization of transducible peptides in Fig. 3, after 15 min of peptide incubation, the medium was removed, and the cells were washed twice with the HBSS buffer. For subsequent confocal image collection, the cells were kept in medium or buffer. To study the effect of the peptide on muscle contraction and intracellular Ca2+ (Fig. 4), cardiomyocytes were electrically stimulated (see below) to permit the access of the peptides to all actin molecules with potential binding sites. During confocal image collection, the electric stimulation was temporarily switched off.
Fig. 1Peptide uptake and intracellular distribution in living adult cardiomyocytes. To study the uptake capacity of different compounds into primary rat cardiomyocytes, different fluorescent compounds were directly added for 1 h to the medium or buffer of freshly isolated cardiomyocytes. After the incubation period, confocal images of the cells were taken without exchange of the medium and after removal of the fluorescent solution and exchange against the fresh medium. Internalization into healthy adult cardiomyocytes occurred after application of 10 μM of the transducible peptides FITC-TAT shown at low (a) and high magnification (b) and TAMRA-TAT-p21 shown at low (c) and high magnification (d) but not with application of 10 μM of a basic control peptide PTD4 (e). Moreover, 0.5% of the vital dye trypan blue (f) or 10 μM of the TAMRA-fluorophore (g) itself were not able to enter healthy cardiomyocytes (rod-shaped cells in culture). Scale bar, 10 μm. Stars indicate the nuclei and arrowheads the nucleoli (N) in the higher magnification images. PC Phase contrast, NP nucleoplasm
Microscopy, image acquisition, and analysis Confocal images were acquired with a Zeiss laser scanning microscope LSM510 Meta mounted on an Axiovert 200M inverted microscope using a 63× phase-contrast oil immersion plan-apochromat objective NA1.4 or a 100× phase-contrast oil immersion plan-neofluar objective NA1.3. For all settings, the main beam splitter was HFT UV/488/543/633, and the specific parameters for the single fluorophores were: FITC, excited at 488 nm light, detected with a 500–530-nm bandpass filter; TAMRA or rhodamine excited at 543 nm, detected with 565–615 bandpass filter; and trypan blue, excited with 633 nm, detected with 650 longpass filter. Phase-contrast images were recorded with excitation at 488 nm and detection in the transmission channel. Laser power for observation was typically 1–5% (488 nm, 25 mW) and 50–60% (543 nm, 1 mW) unless otherwise indicated. Settings were adjusted in a way that image pixels were not over- or underexposed with the range indicator function in the Zeiss LSM image acquisition and examiner software version 3.2.
Measurement of cardiomyocyte shortening and Ca2+ transients Attached cardiomyocytes were washed with HBSS. Cells were loaded with Fura-2-AM for 30 min at room temperature in the dark. The dye solution was removed, and cells were left on HBSS for another 15 min. Only cardiomyocytes of optically intact rod-shaped morphology with clear cross-striation were analyzed. Cardiomyocytes were electrically stimulated until a stable steady-state contraction and Fura-2 signal could be monitored.
Cell shortening and Fura-2 signals were simultaneously measured at 30°C on an Ionoptix Contractility and Fluorescence System (Ionoptix). Cardiomyocytes were electrically stimulated with bipolar pulses of 5 ms duration at 1 Hz. Cell shortening, expressed as percentage of resting cell length, was measured using the video-edge technique at a sampling rate of 240 per second. Ca2+ transients were monitored as ratio of fluorescence emission at 510 nm was obtained by alternate excitation at 340 and 380 nm (340/380 ratio). Data files from 15 consecutive beats recorded at intervals were averaged for analysis. Subsequently, the cardiomyocytes were incubated with the peptides for 15 min without electrical stimulation. The peptide was then removed by replacing the peptide-containing HBSS buffer with normal HBSS buffer without the peptide. The cardiomyocytes were subsequently electrically stimulated, and both the shortening and Fura-2 signals were simultaneously recorded. The effect of the peptide on shortening amplitude and the Fura-2 signal was expressed in percent change compared to the steady-state signals obtained in the preincubation period.
Results and discussion
We recently demonstrated by live-cell confocal microscopy that the TAT CPP was able to shuttle fused peptides into primary blood cells and subsequently modulated their functional properties [10]. In this study, we tested whether TAT CPP could penetrate and if a peptide derived from the N terminus of VLC-1 fused to TAT is able to modulate the contractile function of primary adult cardiomyocytes.
Different basic peptides were incubated with freshly prepared cultures of cardiomyocytes, and peptide internalization was analyzed live by laser-scanning confocal microscopy. The fluorescently labeled minimal transduction domain TAT CPP of the transactivator of transcription from HIV-1 was readily taken up into cardiomyocytes and displayed a strong affinity to the nuclear compartment (Fig. 1a). It could also be visualized in a punctate pattern throughout the entire cardiomyocyte (Fig. 1a). To determine whether the TAT CPP is able to shuttle an attached peptide into cardiomyocytes, we used the TAMRA-labeled TAT-p21WAF/CIP (TAT-p21) fusion peptide [33], which in addition to the transducing moiety contains 26 amino acids derived from the C terminus of human p21WAF/CIP protein. The uptake of this cargo peptide (assessed by appearance in nucleoli) occurred on a timescale of 5 to 10 min after peptide application. Its pattern of localization inside the cardiomyocytes was similar than that seen for TAT alone (Fig. 1c). Basic amino acid residues are a prerequisite for the transduction ability of CPPs [13, 25, 35], and peptides containing a minimum of six basic amino acids have been shown to traverse biological membranes [5, 18]. As a nontransducing control peptide, we chose the TAMRA-labeled peptide PTD4, which contains three arginines in total [23, 33]. More than 1 h after application of this peptide to the cardiomyocyte culture, none of the healthy intact cardiomyocytes took up the fluorescent peptide (Fig. 1e). Similar results were obtained for the vital dye trypan blue (Fig. 1f) and the TAMRA fluorophore (Fig. 1g). Approximately 5% of cardiomyocytes in the cultures were dead or harmed (visible in the phase-contrast images as not rod shaped and with no striations). These cells were flooded with both the CPPs as well as any other control dyes (Fig. 1c,e–g). The cell-permeable TAT-derived peptides displayed a similar enrichment in the nuclear compartment with strong accumulation inside nucleoli (arrowheads) as can be seen in the higher magnification images (Fig. 1b,d). This intranuclear distribution is basically determined by the TAT moiety, which includes a nuclear localization sequence [22, 29, 33].
Having shown that CPP TAT was able to shuttle fused peptides into living cardiomyocytes, we designed a transducible peptide comprising the actin-binding element of the ventricular isoform of human MLC-1 (VLC-1), i.e., the N-terminal residues 1–15 (VLC-1 peptide) connected to TAT, termed VLC-1-TAT (Fig. 2). The hearts of transgenic animals harboring minigenes encoding for the VLC-1 peptide revealed enhanced myosin motor activity and positive inotropic effects [21]. A possible molecular mechanism for the increase in myosin motor activity and contractility is illustrated in the scheme in Fig. 2. MLC-1 isoforms of vertebrate striated muscle play a regulatory role in myosin motor function: The N-terminal part of MLC-1 contains a sticky element (SE), which consists of several positively charged amino acids that bind to the C terminus of actin [3, 6, 16, 31]. The 46 N-terminal amino acids of MLC-1 have previously been modeled on the basis of contemporary crystallographic data [2]. Interactions between the sticky MLC-1 element and actin serve as a negative modulator and decrease the activity of the myosin motor domain. Intracellular delivery of a synthetic sticky MLC-1 element by TAT-mediated transduction therefore would antagonize MLC-1/actin interaction. We predict this would increase myosin motor activity and enhance the shortening amplitude without change of the systolic free Ca2+ activation level of intact cardiomyocytes.
Fig. 2Molecular hypothesis for the enhancement of cardiomyocyte contractility by the cell-permeable VLC-1 peptide. a Primary sequence of VLC-1-TAT peptide color-coded orange for the VLC-1- and yellow for the TAT-moiety. b Schematic sliding filament model displaying actin in violet and the MHC in red. The higher magnification shows the catalytic domain (CD) and the neck domain (ND) of one molecule of the MHC. MLC proteins are colored green with the N-terminal SE inside the essential MLC-1 protein highlighted in red. Upon intramolecular conformational changes in the CD of MHC, the ND swings out, exposing the SE of MLC-1 to an actin molecule in direction to the Z-line. Treatment of cardiomyocytes with the transducible TAT peptide (yellow ball) fused to the SE-peptide (orange) blocks potential binding sites of the native MLC-1 N terminus. After the MHC rearrangement, the MLC-1 protein can no longer interact with actin and downregulate the myosin motor activity. The scheme is based on the pre- and postpower stroke model [19] developed with respect to crystallographic data on the Dicytostelium myosin motor domain [15, 32]
To first test whether the VLC-1 peptide itself might be taken up by isolated cardiomyocytes because of its basic charge, VLC-1 and VLC-1-TAT were applied for 15 min to freshly isolated cardiomyocytes under resting conditions or, in another set of experiments, constant electric stimulation (1 Hz, 20 V). After the fluorescent peptides were washed away, the cells were analyzed live by confocal laser-scanning microscopy (Fig. 3a). VLC-1 alone was not internalized into healthy cardiomyocytes and stained only compromised cells (upper panel, Fig. 3b). To ensure that low concentrations of intracellular VLC-1 were not missed, the laser intensity was increased to its maximum, and still no fluorescent signals were measured (lower panel, Fig. 3b). In contrast, VLC-1-TAT reached the intracellular compartments of more than 95% of adult cardiomyocytes (Fig. 3c).
Fig. 3Transduction and intracellular localization of VLC-1-TAT in isolated adult cardiomyocytes. a Scheme for the application of the VLC-1-derived peptides and confocal imaging. Peptides were applied at a concentration of 1 μM, incubated for 15 min to freshly isolated cardiomyocytes. After removal of the peptide solution, the cells were washed twice and kept in buffer or medium for direct live-cell confocal imaging microscopy. Subsequently, the transduced cardiomyocytes were electrically stimulated with 1 Hz for maximally 1 h. b The negative control peptide TAMRA-labeled VLC-1 stained only dead cardiomyocytes (low magnification, upper panel) but failed to get internalized into healthy cardiomyocytes even when 100% laser power was used to detect potentially weak signals (lower panel). c Application of TAT-VLC-1 to living cardiomyocytes led to rapid internalization of the peptide (upper panel). The lower panel shows transduced VLC-1-TAT at higher magnification in an overlay of the phase contrast and the TAMRA-fluorescence. The arrow indicates the direction of the intensity profile of gray and red intensities in the line scan for the localization analysis of VLC-1-TAT, which was present at sarcomeric structures with enrichment at the actin-containing I-bands (light bands in phase-contrast image)
Unlike the localization of the TAT and TAT-p21 peptides (Fig. 1), the VLC-1-TAT was not detected in the cell nucleus. Detailed analysis of the intracellular distribution of transduced VLC-1-TAT and correlation of the fluorescent signals with the phase contrast images (merge image, lower panel, Fig. 3c) demonstrated that the peptide associates with sarcomeric structures. It showed strong enrichment at the I-bands representing the nonoverlapping zones of the thin actin filaments with the thick myosin filaments (linescan, lower panel, Fig. 3c). After transduction, the VLC-1-TAT peptide remained inside the sarcomeric compartments of the cardiomyocytes for at least 1 h after transferring the loaded cardiomyocytes into the peptide-free buffer solution.
Using the same incubation protocol, we studied the effects of the cell-permeable VLC-1 peptide on muscle function by simultaneously monitoring the shortening amplitude and the systolic and diastolic cytoplasmic Ca2+ fluctuations (measured by Fura-2 fluorescence) of paced adult rat cardiomyocytes (Fig. 4a). As shown in Figs. 4b and c, 1 μM, but not 0.5 μM, of VLC-1-TAT significantly increased the shortening amplitude of adult cardiomyocyte, while the cytosolic Ca2+ signal remained unchanged. The same concentrations of control TAT peptide alone did not change the contractile state or the Fura-2 signals if compared to the basal state during the preincubation period. Higher concentrations of both control and test peptides induced deleterious effects on the cardiomyocyte contraction. It is interesting to note that not only the maximal amplitude of shortening rose but also the maximal rate of shortening and the maximal relaxation velocity increased significantly. Because the Fura-2 signal remained unchanged, the selective effect of the VLC-1-TAT peptide on the contraction parameters suggests a selective and direct effect on the function of the myosin cross-bridges. The Ca2+-sensitizing effect of the VLC-1-TAT peptide could be predicted from the mechanism of action, as the inhibition of the MLC-1/actin interaction may rise selectively myosin motor activity and therefore muscle contraction at a given activating free Ca2+ concentration, i.e., an inotropic effect without additional recruitment of Ca2+. It should be noted that the VLC-1-TAT peptide left the intracellular Ca2+-handling system unchanged, as seen by the fact that the kinetic parameters of the Fura-2 fluorescence signals remained normal (Fig. 4c).
Fig. 4Functional effects of cell permeable VLC-1-TAT in living adult cardiomyocytes. a Original registration of cell length (top) and cytosolic Ca2+ signals (Fura-2 signal, expressed as ratio R between 340 and 380 excitation; bottom) of electrically paced adult rat cardiomyocytes before (basal) and after incubation with 1 μM VLC-1-TAT or 1 μM TAT as control. b Statistical evaluation of the effects of different concentrations of transducible peptides (0.5, 1 μM) on fractional shortening (FS; given in percent of basal contraction obtained before incubation with the peptide), maximal rate of FS (+dFS/dtmax), and maximal relaxation rate (-dFS/dtmax; both given in percent of basal values obtained before peptide incubation) of control peptide (TAT) and VLC-1-TAT. c Statistical evaluation of the effects of different concentrations of transducible peptides (0.5, 1 μM) on peak Fura-2 fluorescence signal (expressed as ratio R between 340 and 380 excitation, given in percent of basal R340/380 obtained before incubation with the peptide), maximal rate of R340/380 (+dR340/380/dtmax), and maximal rate of fluorescence decline (-dR340/380/dtmax; both given in percent of basal values obtained before peptide incubation) of control peptide (TAT) and VLC-1-TAT. Values are means ± SEM, n = 6–9 different cardiomyocytes at each peptide concentration used. Double asterisk, p < 0.01; triple asterisk, p < 0.001 (t test for paired values)
In summary, we demonstrate by live-cell microscopy that CPPs and CPPs fused to peptides were taken up by adult cardiomyocytes with high efficiency and localize to their targets inside the cytoplasm and/or nucleus. The cell-permeable peptide VLC-1-TAT accumulated in the actin-containing I-band of the sarcomeres and was able to enhance the contractility of isolated adult cardiomyocytes without changing the myoplasmic Ca2+ levels. Importantly, the fact that VLC-1-TAT did not affect intracellular Ca2+ concentration and that it only has targets in striated muscle cells makes this peptide uniquely suited as a novel potential therapeutic tool to modulate heart function. VLC-1-TAT is a powerful new drug candidate to improve the contractile state of the failing heart.

cardiomyocytes
ventricular light chain-1 (vlc-1)
peptide transduction
cell penetrating peptide (cpp)
drug delivery


Identification of elderly fallers by muscle strength measures

For efficient prevention of falls among older adults, individuals at a high risk of falling need to be identified. In this study, we searched for muscle strength measures that best identified those individuals who would fall after a gait perturbation and those who recovered their balance. Seventeen healthy older adults performed a range of muscle strength tests. We measured maximum and rate of development of ankle plantar flexion moment, knee extension moment and whole leg push-off force, as well as maximum jump height and hand grip strength. Subsequently, their capacity to regain balance after tripping over an obstacle was determined experimentally. Seven of the participants were classified as fallers based on the tripping outcome. Maximum isometric push-off force in a leg press apparatus was the best measure to identify the fallers, as cross-validation of a discriminant model with this variable resulted in the best classification (86% sensitivity and 90% specificity). Jump height and hand grip strength were strongly correlated to leg press force (r = 0.82 and 0.59, respectively) and can also be used to identify fallers, although with slightly lower specificity. These results indicate that whole leg extension strength is associated with the ability to prevent a fall after a gait perturbation and might be used to identify the elderly at risk of falling.
Introduction
One-third of those over 65 years of age fall at least once per year. About 50% of them will suffer recurrent falls (Masud and Morris 2001). Even in the large group of relatively fit and healthy elderls, falls are common (Stel et al. 2003a). Consequences of falls are known to contribute substantially to the prevalence of health problems (Lord and Dayhew 2001), health care costs (Stevens et al. 2006), and to lost quality of life (Cumming et al. 2000). Prevention of falls is therefore imperative and should address the people at risk and the physiological causes of falling (Lord et al. 2003).
Numerous epidemiological studies have shown that falls are associated with many risk factors (for overviews see e.g., Lord et al. 2001). Reduced muscle strength, especially of the lower limbs, has been indicated as one of the most important risk factors for falls (Lord et al. 2003; de Rekeneire et al. 2003; Moreland et al. 2004; Rao 2005; Rubenstein 2006; Skelton et al. 2002). Experimental studies can provide causal verification and insight into the contribution of muscle strength to fall incidence in standardized situations. For example, tripping over an obstacle, which accounts for up to 60% of falls (Berg et al. 1997; Roudsari et al. 2005), can be induced and measured in older adults in experimental settings (Pavol et al. 1999; Pijnappels et al. 2005c; Schillings et al. 2005). Previous findings provided insight into the (neuro)physical requirements of preventing a fall after tripping over an obstacle (for overviews see van Dieën et al. 2005; Pijnappels et al. 2007). It was shown that older adults were less able to successfully recover balance than younger adults, due to lower maximum ankle moments and lower rates of moment generation around all leg joints during the push-off phase of balance recovery (Pijnappels et al. 2005c). These results suggest that leg strength may be the limiting factor in preventing a fall.
Pavol et al. (2002) have investigated the relation between muscle strength and the outcome of an induced trip. In a group of older adults, they compared isolated ankle, knee, and hip flexion and extension strength between those who recovered from a trip, and those who fell. Although some strong adults fell, presumably due to their high walking velocity, they also found that muscle strength was lower in several other fallers.
If low muscle strength indeed is a physiological cause of falls, this raises the question whether people with a high risk of falling can be identified by means of relatively simple maximum muscle strength measures. The aim of this study was to find the best predictor of falls after a gait perturbation in a standardized situation, from a range of muscle strength measures in older adults.
There are numerous maximum strength capacity measures that have been assessed in aged populations, for example to evaluate training interventions (Ferri et al. 2003; Reeves et al. 2005b; Verschueren et al. 2004) or to compare fallers with non-fallers (Gehlsen and Whaley 1990; Pavol et al. 2002). We focused on the most common measures by use of a dynamometer, isometric ankle and knee extension moment and rate of moment generation. In addition to isolated joint moments, total lower limb push-off force can be measured by a leg press dynamometer (Gehlsen and Whaley 1990). Performance on a maximum vertical jump could be a more functional test used to assess maximum lower limb strength (Izquierdo et al. 1999; Runge et al. 2004). Finally, hand grip strength is thought to reflect general body strength and has been used as predictor of falls in epidemiological studies (Pluijm et al. 2006).
Elderly volunteers performed these maximum strength capacity tests and we measured their ability to prevent a fall after being tripped. We hypothesized that maximum muscle strength measures can be used to differentiate fallers from non-fallers. If so, this would not only allow identification of potential fallers, but also indicate specific limiting factors to target with exercise-based interventions.
Methods
Seventeen healthy older adults participated [10 women: age 71 (SD 4.5) years, mass 75 (SD 9) kg, height 1.68 (SD 0.09) m]. All participants were fit and had no orthopedic, neuromuscular, cardiac or visual problems. The Ethics Committees the VU Medical Center and of the Manchester Metropolitan University approved the procedure and all subjects gave their written, informed consent before participation.
Capacity measures
Participants performed a series of static and dynamic maximum force generating capacity tests. First, isometric ankle plantar flexion moment and knee extension moment (maximum as well as rate of moment development) were assessed using a dynamometer. In view of constraints on equipment and subject availability, nine of the participants were tested using an isokinetic dynamometer (Cybex Norm, New York, USA) (Reeves et al. 2004) and eight using a custom-built dynamometer (de Ruiter et al. 2004) (Fig. 1a, b). Isometric knee extension moment was measured in both settings with a knee joint angle of 90° (0° full extension) and a hip angle of 85° (0° supine position). Isometric plantar flexion moment was measured using the Cybex with the knee in full extension and the ankle at 0° (anatomical position). In the custom ankle dynamometer, the knee joint was fixed at 90° and to obtain a similar gastrocnemius muscle length compared to that during the tests using the Cybex, the ankle angle was set at 20° dorsiflexion (see equation in Reeves et al. 2005a, p. 2280). Plantar flexion moment of the right leg (push-off leg in the tripping experiments) and knee extension of the left leg (landing leg for tripping) were measured. Measurements resulted in comparable values between settings and were repeated 3 times with 3 min rest between contractions. Subjects first performed a maximum isometric contraction by increasing their effort in a linear ramp fashion, so that maximum moment was reached within 2 s and then maintained for 3–4 s. From these measurements, the maximum moments (anklemax and kneemax) were calculated, relative to body mass. Next, subjects were instructed to increase their moment from 0 to at least 70% of their maximum as quickly as possible, without counter movement. Moment onset (2 N m above baseline) was determined and rate of moment development (anklertd and kneertd) was calculated over the first 100 ms after moment onset and expressed relative to body mass.
Fig. 1Pictures of the experimental settings for a right plantar flexion moment measurements, b left knee extension moment measurements, c leg press push-off force measurements, d jumping measurements, and e tripping experiments
Maximum leg press push-off force of each lower limb was measured for all subjects using the same leg press fitness instrument (MR Systems, Haarlem, the Netherlands) (Fig. 1c). Subjects performed a one-legged (horizontal) push-off, starting with the ankle joint angle at 0° and the knee joint angle at 30°. This position was comparable to the configuration of the support limb during tripping (Pijnappels et al. 2005a, c). The fore-foot was positioned on a 10 × 10 cm force transducer (AMTI M3-1000, Watertown, USA) that was mounted in the push-off platform. Subjects first performed a maximum isometric contraction with the sled fixed and subsequently a maximum dynamic contraction with the weight stack set equal to body mass, so the only external resistance was the mass and inertia of the body and sled. Both conditions were repeated 3 times for both legs with a 3 min rest between contractions. The maximum horizontal leg press push-off force (legpressmax) was calculated from the isometric contractions and the rate of force development (legpressrfd) was calculated from the dynamic contractions over 100 ms after onset of force generation, averaged over both legs and expressed relative to body mass.
For the jumping measurement, subjects performed a maximum two-legged counter movement jump (Fig. 1d). Position data of markers placed bilaterally on the trochanter major were measured by Optotrak cameras (Northern Digital, Waterloo, Canada). From the best of two attempts, maximum jump height (jumph) was calculated as the vertical distance between the highest averaged hip position during jumping and averaged hip height during normal standing.
Hand grip strength was measured using a digital strain-gauge dynamometer (Takei TKK 5401, Takei Scientific Instruments, Tokyo, Japan). Subjects performed two maximum force trials with each hand. The maximum values of the right and left hand (in kg) were added together (handf).
Tripping measurements and falls
The ability to prevent a fall after a gait perturbation was determined by tripping measurements (Pijnappels et al. 2005c). Subjects walked at a self-selected velocity over a 12 m walkway. Fourteen 15 cm high obstacles were hidden at the left side and seven obstacles were hidden at the right side of the walkway, over a total distance of 1.5 m (Fig. 1e). In 2–5 of 40 trials, one of the obstacles suddenly appeared (based on online kinematic data) to catch the subject’s left swing leg at mid-swing, usually causing an elevating strategy for balance recovery. This strategy involves the limb contacting the obstacle being elevated over the obstacle (Eng et al. 1994; Schillings et al. 2000), whilst the contralateral support limb provides push-off. Subjects were aware that they could be tripped on either leg, but did not know whether and where an obstacle would appear in the ongoing trial. They wore a safety harness that moved along a track above the walkway. Subjects were classified as fallers based on visual detection of full body support by the harness (checked by video), in more than 50% of the tripping trials.
Statistical analysis
Pearson’s coefficients of correlation between the capacity measures were calculated. In case of high correlations between the dependent variables, as we found, the power of a multivariate analysis of variance (MANOVA) decreases when the expected effect sizes are reasonably large and consistent in the same direction (Cole et al. 1994). We therefore focused on separate univariate ANOVAs to determine which capacity measures differed between fallers and non-fallers. The variables that appeared to be determinants of falls from the ANOVAs were used in discriminant analyses, in order to find the variable(s) best discriminating between fallers and non-fallers and to determine sensitivity and specificity. In addition, a cross-validation was performed to assess the generalizability of the predictive models; each case was classified by the functions derived from all other cases (leave-one-out-classification). SPSS statistical software was used and the level of significance was set at p < 0.05.
Results
Tripping measurements and falls
Seven of the participants (all women) were fully supported by the safety harness in more than half of the tripping trials. Six of them fell in all trials; the other fell in three out of five trials. Non-fallers were never fully supported by the safety harness. The averaged walking velocity was 1.38 (SD 0.17) m/s and did not differ between fallers and non-fallers (p = 0.488).
Capacity measurement as predictor for falls
legpressmax, jumph, and handf were significantly correlated with almost all other capacity measures (Table 1). Leg press push-off force was not significantly different between the left and right leg of the subjects (p = 0.496 for legpressmax and p = 0.149 for legpressrfd), there was no significant interaction with groups (p = 0.133 for legpressmax and p = 0.919 for legpressrfd), and the values were highly correlated between legs (overall, r = 0.813, p < 0.001 for legpressmax and r = 0.829, p < 0.001 for legpressrfd).
Table 1Correlation coefficients between capacity measuresanklertdkneemaxkneertdlegpressmaxlegpressrfdjumphhandfanklemax0.63b0.230.320.76b0.370.61b0.40anklertd10.360.70b0.68a0.400.65b0.51akneemax10.72b0.53a−0.020.340.71bkneertd10.57a0.360.55a0.78blegpressmax10.330.82b0.59alegpressrfd10.290.23jumph10.69bSignificant correlations are in bold.ap < 0.05bp < 0.01
All but one of the univariate ANOVAs resulted in significant differences, indicating several measures to be different between fallers and non-fallers (Fig. 2). Ankle and knee capacity measures (both maximum and rate of moment development), maximum leg press force, jump height, and grip strength were significantly lower for fallers compared to non-fallers.
Fig. 2Averaged group differences in capacity measures between fallers and non-fallers. Maximum (max) and rate of moment development (RTD) of plantar flexion moment (anklemax and anklertd) and knee extension moment (kneemax and kneertd), maximum and rate of force development (RFD) of leg press push-off force (legpressmax and legpressrfd), jump height (jumph), and hand grip strength (handf). Significant differences between groups are indicated with p-values
These seven variables were entered in a stepwise discriminant analysis, which resulted in a significant model (p = 0.001) with legpressmax as the only predictor. This model resulted in 94% correctly classified cases and a cross-validation of 88% (Table 2). For comparison, discriminant analyses with the ankle and knee capacity tests (stepwise) and with jump height and grip strength as single predictors also resulted in significant predictive models, with slightly lower predictive values (Table 2). Figure 3 presents the distribution of individual jumph,, and handf values, both in relation to legpressmax.
Table 2Predictive variables resulting from stepwise discriminant analyses and cross-validation on capacity measuresCapacity measuresPredictive variablepDiscriminant analysis (sensitivity/specificity%)Cross-validation (sensitivity/specificity%)legpressmax, anklemax, anklemax, kneemax, kneertd, jumph, handflegpressmax0.00186/10086/90aanklemax, anklertd, kneemax, kneertdanklemax, kneemax0.00771/9071/90jumphjumph0.00286/9086/80handfhandf0.00386/8086/80aBest predictive modelFig. 3Distribution of the individual values of maximum leg press push-off force (legpressmax) with jump height (jumph) and hand grip strength (handf) for fallers and non-fallers. Crosses indicate the incorrect classified cases (x for legpressmax, + for jumph and handf)
Discussion
It is important to identify individuals most at risk of falling, because they should be considered with priority for receiving targeted exercise interventions aimed at reducing the incidence of falls. This study investigated the possibility to identify fallers from maximum strength measures that could be applied in clinical settings. The results showed that participants who fell after a gait perturbation could indeed be identified based on these measures. Especially, a classification model based on maximum leg press push-off force yielded high sensitivity and specificity in cross-validation.
The subjects in this study were relatively fit and healthy older adults. Nevertheless, muscle strength varied greatly between subjects, with coefficients of variation between subjects ranging from 23 to 60% for all strength measures. Pavol et al. (2002) found that not only people with low muscle strength, but also the stronger people had a higher risk of falling after a trip. This was explained by a higher walking velocity in the stronger group, which increases the demands for adequate balance recovery. These subjects mainly used a lowering strategy, which implies that they were tripped at late swing (Eng et al. 1994; Pijnappels et al. 2005b). Since the trip was induced manually, it may be that faster walkers were more likely to be tripped in late swing. It is therefore unclear whether velocity or instant of trip initiation was responsible for a number of stronger subjects falling. In our study, however, walking velocity was not different between fallers and non-fallers and in the range of “fast walkers” of the study of Pavol, even though we did not standardize it and explicitly instructed subjects to walk at their normal and comfortable velocity. Furthermore, we were able to trip all subjects at exactly mid-swing (when the toe height is minimal and a trip is most likely in daily life). This implies that the effect of the trip by impact with the obstacle was equal over subjects and could not affect the outcome of the trip. Yet, this also may suggest that instant of trip initiation and in a more heterogeneous group, differences in preferred walking velocity must be taken into account when attempting to identify fallers.
Although we were able to trip our subjects repeatedly, three of the fallers and one non-faller did not complete the whole protocol up to five tripping trials, due to discomfort. Nevertheless, the fallers fell in all trials and the non-faller did not fall in four tripping trials. Hence, the number of tripping trials did not affect the classification of the participants.
Hip extension and knee flexion strength were not measured, but might also contribute to fall prediction, as the rate of development of hip extension and knee flexion moments during push-off after tripping were found to be lower in older fallers compared to young adults (Pijnappels et al. 2005c). Since especially hip extension capacity is quite difficult to determine (especially in a clinical setting) and given limitations in the total number of capacity measurements to be performed in a single session, these variables were not included in the present study.
During the push-off phase of a tripping reaction, the knee is generating an internal flexion moment while extention (Pijnappels et al. 2005a). Preliminary data suggest that a high extension moment is required in the leg making ground contact at landing (van Dieën et al. 2005) and a limited capacity to generate sufficiently high knee extension moments might thus increase the fall risk. In this study, fallers could not position their recovery foot adequately and were therefore fully supported by the safety harness, mostly even before landing. This underlines that most of the balance recovery takes place during push-off by the support limb. However, as falls can also occur after landing (Pavol et al. 2001), the requirements for landing need further investigation.
Fallers and non-fallers were classified in this study based on their ability to prevent a fall after an experimentally induced gait perturbation. In daily life, there are many various ways in which people fall. Furthermore, this experimental study included a small number of participants, which might have influenced the predictive values. A prospective study with a larger cohort is necessary to generalize the results of this study to the prediction of falls in daily life.
Overall, sufficient leg extension strength is required for adequate balance recovery after tripping. This might explain why a capacity measure that involves extension of all lower limb joints (i.e., leg press push-off) yielded the best identification of fallers. This one-legged task is highly similar to the actual movement during the push-off phase of tripping (Pijnappels et al. 2005a, c).
Jumping also requires rapid moment generation and was therefore expected to be a good and practical measure of whole lower limb strength. Studies have shown that maximum jumping forces are lower in the elderly (Izquierdo et al. 1999; Runge et al. 2004) and related to balance performance (Izquierdo et al. 1999). Jump height indeed resulted in good sensitivity and specificity. This task was less similar to the actual tripping action than the leg press and does not solely reflect strength, as it also requires complex coordination of many joints. One might argue that the older fallers, who were all women, may have been more afraid to jump maximally than their counterparts. However, jump height was strongly correlated to maximum leg press push-off force, which was measured in a secure and comfortable setting. Jumping performance, therefore, more likely reflects lower limb strength than an effect of gender or fear.
Fall incidents and the ensuing injury process are multi-factorial. Muscle strength is only one of many factors that associate with falls and that has to be taken into account when identifying one's fall risk. Yet, this particular factor has been indicated as one of the main causal factors for falls and is essential in risk profiles. For example, in the physiological profile approach by Lord et al. (2003) muscle force is tested in lower extremity muscle groups. However, these tests are not based on empirically demonstrated causal relations. Our experimental study allowed investigating the relation between strength measures and gait perturbations in a standardized situation. It can be questioned whether the findings in this study on tripping over an obstacle in mid-swing can be transferred to other fall mechanisms that occur in daily life. Yet, leg extension force has also been indicated to play an important role in other fall mechanics, such as slipping (Chambers and Cham 2007; Tang and Woollacott 1999). The results of this study may therefore contribute to improvement of risk profiles for identification of high-risk fallers, particularly by using relatively simple but specific measures of maximum strength.
For clinical use, a maximum vertical jump test might be more practical than the leg press test, as it requires little instrumentation. Rittweger et al. (2004) demonstrated that such a test is feasible also for subjects over the age of 80 and that reliability is high.
Hand grip strength is a common clinically used strength capacity measure. It has been shown in epidemiological studies to be related to leg extension strength and to be associated to recurrent falling in daily life (Stel et al. 2003b). Our data indeed showed that grip strength is significantly correlated with lower limb capacities and that it can be used to identify fallers from non-fallers. Although hand grip strength is easy to measure and assumed to be related to whole body strength, one should bear in mind that this measure may be less accurate as a predictor for falls it is not task specific for balance recovery.
With respect to fall prevention, the associations found in this study suggest that maximum whole leg extension force is an important target in conducting exercise-based interventions. In a pilot study, we showed that lower limb resistance training has the potential to improve balance recovery, as older trainers improved more in moment generation upon tripping than controls (Pijnappels et al. 2007). It can be questioned whether fall prevention training should focus on strength training alone, as task-specific training of motor skills has been shown to be important (Bieryla et al. 2007; Owings et al. 2001; Troy and Grabiner 2005). A combination of both strength and balance training is most likely to be most beneficial for reduction of fall incidence (Gillespie et al. 2003). Finally, it should be noted that training effects are only valuable if task-specific requirements can be achieved and it can be questioned whether this is the case in older and more frail elderly. Therefore, resistance training may be particularly useful in the group of relatively fit and healthy elderly (Barry and Carson 2004; Hunter et al. 2004; Latham et al. 2004; Macaluso and De Vito 2004), which might prevent these people from becoming recurrent fallers.
Conclusion
Relatively simple and accessible measures of maximum strength did identify elderly fallers from non-fallers after a standardized gait perturbation. The capacity to generate maximum extension force by the whole leg (e.g., in a leg press apparatus or during jumping) resulted in the best classification of older fallers and non-fallers. Follow up studies on larger cohorts with a wide range of muscle strength and walking velocities are necessary to generalize these results towards a valid prediction of fall risk.

perturbation
classification
ageing
fall risk
physical capacity


Clinical effect of continuous corrective force delivery in the non-operative treatment of idiopathic scoliosis: a prospective cohort study of the triac-brace

A prospective cohort study of skeletally immature idiopathic scoliotic patients treated with the TriaC brace. To determine if the TriaC brace is effective in preventing curve progression in immature adolescent idiopathic scoliotic patients with a very high risk of curve progression based on reported natural history data. The aim of the newly introduced TriaC brace is to reverse the pathologic transverse force pattern by externally applied and continuously present orthotic forces. In the frontal plane the force system used in the TriaC brace is similar to the force system of the conventional braces. However, in the sagittal plane the force system acts only on the thoracic region. In addition, the brace allows upper trunk flexibility without affecting the corrective forces during body motion. In a preliminary study it is demonstrated that the brace prevents further progression of both the Cobb angle and axial rotation in idiopathic scoliosis. Skeletally immature patients with idiopathic scoliosis with curves between 20 and 40° were studied prospectively. Skeletally immature was defined as a Risser sign 0 or 1 for both boys and girls, or pre-menarche or less than 1-year post-menarche for girls. Curves of less than 30° had to have documented progression before entry. The mean age of the patients at the start of treatment was 11.3 ± 3.1 years. All measurements were collected by a single observer, and all patients were followed up to skeletal maturity. Treatment was complete for all participants when they had reached Risser sign 4 and did not show any further growth at length measurements. This was at a mean age of 15.6 ± 1.1 years, with a mean follow-up of 1.6 years post bracing. In our study a successful outcome was obtained in 76% of patients treated with the TriaC brace. Comparing our data to literature data on natural history of a similar cohort shows that the TriaC brace significantly alters the predicted natural history. The current study demonstrates that treatment with the TriaC brace reduces the scoliosis, and that the achieved correction is maintained in some degree after skeletal maturity is reached and bracing is discontinued. It also prevents further progression of the Cobb angle in idiopathic scoliosis. The new brace does not differ from the conventional braces as far as maintaining the deformity is concerned.
Introduction
The purpose of this paper is to present the results of a prospective study of a new orthotic device for the non-operative treatment of idiopathic scoliosis. Data are provided in a format to facilitate comparison with natural history data.
The main concern in patients with idiopathic scoliosis relates to curve progression and the resulting cosmetic deformity. The risk of curve progression is correlated primarily to periods of rapid skeletal growth [3, 5, 6, 11, 14, 41, 42]. Factors related to growth potential, such as patient age at the time of diagnosis, status of menarche, and Risser sign, have been shown to be important predictors of the progression of scoliosis [3, 15, 29, 39]. In addition to future skeletal growth, curve magnitude and curve shape are further predictors of progression of idiopathic scoliosis. Large initial curves, thoracic curves, and double major curves are more likely to progress [3, 29, 39].
Currently bracing is the accepted nonoperative treatment to prevent curve progression in mild to moderate scoliosis during the growth period. A prospective multicenter study, performed by the Scoliosis Research Society, reported that brace treatment has a significant effect on curve progression of idiopathic scoliosis [36]. However, others doubt the effectiveness of braces [12, 16, 19, 32]. Just as for the natural history of untreated curves, Lonstein and Winter [30] found a relationship between the final outcome of brace treatment and curve factors and factors that predict future skeletal growth. In biomechanical theories describing the patho-physiological mechanism of scoliosis, spinal growth is thought to be the main driving force responsible for curve progression [35, 42, 44, 47, 48].
Throughout history, external devices have been used to correct deformities and immobilize the spine. Brace designs have changed periodically over the years, but most modifications have solely focused on improved efficacy and failed to acknowledge the importance, especially to teenagers, of physical appearance. This age group resists acting or looking different from their peers, which obviously occurs when a visible brace is worn. Modern materials, lower profiles, and reduced wearing times have been tried in attempts to reduce resistance to and the emotional difficulties encountered with wearing braces.
The aim in the design of a new orthosis was to create a system which is at least as effective as the TLSO’s, with improved comfort for the wearer. The newly introduced brace should reverse the transverse force pattern by externally applied and constantly present orthotic forces without limiting the normal body motions of the patient and an increased comfort level for the patient.
Construction and working principle of the TriaC orthosis
The name TriaC is based on the three C’s of Comfort, Control, and Cosmetics. Currently, the orthosis is produced by Somas International, St Anthonis, the Netherlands. For this orthosis, the choice has been made to apply continuous correction forces on the chest with the aim to prevent curve progression during the growth period. A basic requirement for such a brace is that the brace force must be able to follow the main body motions of the patient. To achieve this goal required the use of a flexible coupling, connecting the thoracic and lumbar parts of the orthosis. The thoracic part and lumbar sections each generate their own corrective force on the body of the patient. Both parts are connected to each other by a flexible coupling that is placed on the opposite side of the thoracic force pad (Fig. 1). This coupling enables the patient to bend forward, backward and sideways while the correction forces are maintained (Fig. 2). The thoracic force pad is located just below the shoulder blade and applies a corrective force in a lateral–anterior direction. The lumbar force pad is placed between the pelvis and the lower ribs and acts on the lumbar muscles in a lateral direction. A third counter force on the hip region ensures that the orthosis is in equilibrium.
Fig. 1The TriaC orthosis, with a thoracic part, a lumbar part and a flexible couplingFig. 2The flexible coupling
Placing a flexible coupling between the thoracic and the lumbar part of the orthosis determines the unique force pattern on the patient both with respect to the location of the forces as with respect to the relation between the forces (Fig. 2). Modelling and clinical use have shown that the TriaC is suitable for all curve types, with the exception of curves with an apex at the 12th thoracic and the 1st lumbar vertebra. For these curve type the Lumbar force pad must be placed at the location of the flexible coupling which is not possible in the current construction of the orthosis. The construction as described, makes the orthosis force driven. When the patient moves her or his body in any direction (e.g. flexion, extension, rotation or lateral bending), the force pattern exerted by the orthosis moves along with the patient and thus is maintained constant. For this reason we defined the orthosis as “dynamic” because the orthosis moves dynamically along with the body motions of the patient.
In contrast, conventional orthoses are displacement driven. A rigid shell shaped in the desired corrected position of the patient applies a corrective force only when the thorax of the patient presses against the pads of the brace. When the body of the patient moves away from these pads the rigid brace is not able to follow the body motion of the patient and therefore the brace no longer generates forces on the patient. Therefore, we defined these orthosis as “static”.
Materials and methods
From 1997 to 2005 all consecutive patients who met the inclusion criteria were treated nonoperatively with the TriaC orthosis and studied prospectively. The indication for treatment was a progressive curve with a Cobb angle between 25 and 40°. All curves, except curves already at >30°, had to show documented progression of at least 5°. Patients had to be skeletally immature, defined as a Risser sign 0 or 1 for both boys and girls; in addition, girls are pre-menarche or less than 1-year post-menarche. All the curves had to be flexible as demonstrated by at least a 40% correction on the bending radiograph at the first visit. Patients with the apex of the curve at T12 and L1 were excluded, as were patients with a systemic disease that could possibly influence the outcome of the study (Table 1).
Table 1The in and exclusion criteria for the treatment with the TriaC orthosisInclusion criteria Idiopathic scoliosis with a Cobb-angle between 20 and 40° Skeletally immature  Risser 0–1 status   Pre-menarche   Post-menarche <1 year Primary thoracic apex between the 7th and 11th thoracic vertebra Primary lumbar apex between the 2nd and 5th lumbar vertebraFlexible spinal column as evidenced by at least 40% correction on bending filmsExclusion criteria Idiopathic scoliosis <20° and >40° Other types of scoliosis Skeletal age >Risser 1 Rigid curves Thoraco-lumbar curves with an apex at the 12th thoracic and the 1st lumbar vertebra Patients with a systemic disease which could influence the study parameters
All patients who met the in- and exclusion criteria were treated with the TriaC orthosis. An informed consent form was obtained from all parents of the subjects in regard of the treatment with a new orthosis. According to the medical ethics committee no informed consent was necessary in case of the follow-up schedule because there is no difference with the patients treated with a Boston brace in our clinic.
If during the treatment period the orthosis failed to stop progression and the curve increased significantly, the patient was placed into a Boston Brace. This procedure was chosen to get a first indication whether a Boston brace would be able to prevent curve progression in patients where the TriaC failed.
Failure was defined as either a Cobb angle of >45°, i.e. the generally accepted indication for surgery, or if the curve progressed 5° or more compared with the measurement at study entry [16, 24–31]. During the nonoperative treatment patients were checked by an orthotist at the outpatient clinic every 4 months, where the subjective compliance was documented. At every visit a PA and lateral radiographs were made. The patients were allowed to wean of the braces at skeletally maturity, i.e. when the radiographs showed Risser sign 4 or, for girls, 2 years post-menarche and patients did not show any further growth at length measurements. At every visit standing and sitting height of the patient was recorded as an additional measurement, and these had to show no further increase. Because the TriaC is designed as a dynamic orthosis, it possibly preserves the muscle status of the patient. As a result the weaning process could be faster than with rigid Boston type orthoses. In this study, weaning was generally complete within 4 weeks, whereas for rigid braces this process may take up to one year. After terminating the treatment patients were seen once a year with an AP and lateral X-ray.
Radiographic analysis
All radiographs were obtained in a standing position. The posterior–anterior projection was used to minimize the radiation dose to the breast. All radiographs were made, using a standardized protocol. Anatomical vertebral landmarks are identified and manually marked on a 21 in. high resolution monitor using a pointer [46]. Following the placement of the markers the Cobb angle is calculated, using automatic detection of the apex and end vertebrae [8].
In this article, only the Cobb-angle data are used for comparison with literature data on the natural history.
Results
Out of a total of 212 patients that were treated at the University Hospital of Groningen during the study period, 63, or 30%, met all the in- and exclusion criteria and form the study group. There were seven patients (11%) with a single curve and 56 (89%) with a double curve. The group included six boys and 57 girls. The mean age of the patients at the start of treatment was 11.3 ± 3.1 years. The treatment was complete when the patients had reached Risser 4 with a mean age of 15.6 ± 1.1 years. The mean Cobb angle of the primary curves before the brace treatment was started was 30.2° ± 7.5°. The secondary curves had a mean Cobb angle of 22.3° ±  6.4°.
The distribution of the location of the apex vertebra of the primary curves is shown in Fig. 3.
Fig. 3The distribution of the apex level of the primary curves in the study group
The patient group was divided into a success group and a failure group whereby failure was defined as a Cobb angle of >45° or curve progression of more than 5° compared to baseline [2, 7, 9, 18, 21, 33, 34, 36, 40].
The X-rays showed a mean initial correction of 22 ± 26% for the primary curves, and a mean correction of and 28 ± 35% for the secondary curves. The large variation was primarily caused by the big difference between the initial corrections seen in patients that were considered a success in comparison to patients that were considered a failure. In the success group the initial correction of the primary curve was 34 ± 17% whereas the initial correction in the failure group was −16 ± 17%. For the entire study group the success rate is 76% (48 successes) for a failure rate of 24% (15 failures). In the single curve group the mean Cobb angle at start of the treatment was 35°± 11. The mean initial correction achieved in this group was 23 ± 9%. There were no failures in this group. Although this difference is remarkable compared to the double curved group the single curve group is too small to draw conclusions.
In Table 2 an overview of the success rate is shown, divided per initial Risser sub group (Risser 0 and Risser 1) and divided by the initial Cobb angle at start of the treatment.
Table 2Number of successes/total patients in subgroupsCobb angleSuccess rateRisser 0Risser 120–29°76% (16 of 21 patients)30–39°74% (26 of 35 patients)86% (6 of 7 patients)Total75% (42 of 65 patients)86% (6 of 7 patients)
The actual number of failures in our patient population was 15 out of 63 patients, or 24%. All patients in whom the brace failed went on to surgery, including the nine patients who were also treated with a Boston style brace after the TriaC brace had failed. A more detailed view of the correction of the primary Cobb angle is presented in Fig. 4. In this figure the mean correction percentage of the Cobb angle is shown as a function of the percentage of the total treatment brace period. The treatment period is considered to be at 0% at the start of the treatment and at 100% at the time the brace is discontinued.
Fig. 4Percent correction of the Cobb angle over the entire TriaC treatment period in the success group
It is shown that it was not possible to completely maintain the initial correction for the whole treatment period. At the end of treatment, the mean correction of the Cobb angle in the success patients had decreased to 19 ± 13%. This decrease in correction does not appear to follow a linear pattern. When a relation between the time that the patient wears the brace, and the loss of correction in the brace is assumed the correlation is only (very) weak (ρ = −0.33). The mean correction at the first visit after discontinuation of the brace the treatment was 20 ± 15%. All patients in whom the brace treatment was a success were followed with a mean follow-up of 1.6 years and longest follow-up of 6 years post-bracing. Figure 5 shows the mean correction at different follow-up periods postbracing.
Fig. 5The mean correction during follow-up after termination of successful brace treatment
Because the orthosis does not affect the lateral curvature no significant changes were expected. The analysis of the sagittal curve is displayed in Fig. 6.
Fig. 6Lateral curve during treatment
Discussion
Braces are the oldest recorded method of treatment for spinal injury and deformity. The primary goal in treating patients with scoliosis deformities is to stabilize the curves to prevent further progression of the deformity. Closely related to this is the goal of achieving correction of the spinal deformity, although this is not part of the therapeutic regimen for every patient. Brace treatments do not generally correct the scoliosis, but prevent further progression, i.e. bracing has a “holding effect” [49]. In most published studies, the brace treatment has been considered a failure if the patient needed operative stabilisation or if the curve progressed 5° or more after the initiation of treatment [2, 7, 9, 18, 21, 30, 33, 34, 40]. However, these should not be the only criteria to determine whether brace treatment is successful. The treatment should also improve the patient’s outcome when compared with the expected natural history.
The aim of this study was to investigate whether the TriaC-brace affects the natural history of idiopathic scoliosis. Bunnell [3], studied the natural history of idiopathic scoliosis in a group of patients with similar characteristics to our study group with respect to curve magnitude and age at the first visit. He showed that for the group as a whole 68% show a curve progression of 5° or more and only 34% of the patients showed a curve progression of 10° or more. The latter progression rate is in the same range as the failure rate reported in most of the retrospective studies, regarding overall brace effectiveness. However, the progression rates are not equal for every sub group of scoliotic patients. Lonstein and Carlson [29] reported that patients with Risser 0 or 1 maturity and curves larger than 20° are three times more likely to experience curve progression than those with curves smaller than 20°. For those with curves exceeding 20°, patients with Risser 0 or 1 maturity also are three times more likely to experience curve progression than are patients with Risser 2–5 maturity. They classified Risser 0–1 patients with curves between 20 and 29° as high risk (40–70%) and Risser 0–1 patients with curves between 29 and 39 as very high risk (70–90%). Bunnell [3] also found Risser sign and curve size to be good prognostic factors for curve progression in untreated idiopathic scoliosis. In his series patients with a Risser 0 at the time of diagnosis had a 68% risk of progression 10° or more. This risk was decreased to 52% in those who had a Risser sign of 1 or 2 and was further decreased to 18% for those with Risser 3 or 4. Nachemson et al. [36] reported that 66% of skeletally immature female patients with untreated idiopathic curves between 25° and 35° will experience curve progression greater than 5.
All patients in our study started at Risser 0–1 maturity and had a curve above 30° or, if they had an initial curve below 30° had to show documented progression of ≥5° before enrolment in this study. Wever et al. [47] showed that there is a strong correlation between the curve progression and spinal growth of the patients. Usually the spinal growth is highest around the beginning of the menarche and therefore we only included female patients when they were before or less than 1 year after menarche. By using these inclusion criteria we aimed to include only patients at the highest risk for curve progression.
In order to evaluate whether the TriaC brace alters the natural history of the high-risk patients, we estimated the natural progression rate of our patient group based on the literature as discussed. Our estimates are based on the calculation of a progression factor for all patients in the cohort as suggested by Lonstein and Carlson [29]. According to their algorithm, all our patients individually would have a change of 70–100% of progression of their curves. Based on these data we chose the low end of this range and assumed that in our patient group 70% of the curves would progress without treatment. The actual number of failures in our patient population was 15 out of 63 patients, or 24%, which is a significantly different from the expected number of 44 (P < 0.001).
The validity of the sample size was evaluated by calculating the 95% confidence interval for the failure rate, which was between 11 and 36% (7–23 patients). Thus, the difference in outcomes remains statistically significant. These data show that the TriaC brace significantly altered the predicted natural history of curve progression. The comparison of the results of this study and other brace studies reported in the literature is difficult because of differences in sample size, skeletal maturity and curve magnitudes at the initiation of bracing. Furthermore, some reports excluded those patients who were not compliant with their bracing regimen. These large variations render the value of a statistical comparison of the results doubtful [51]. By applying the inclusion criteria used in this study to the literature data, a more uniform group has been created, making the statistical comparison more reliable. Nachemson et al. [36] prospectively compared 111 adolescent idiopathic scoliotic female patients with curves between 25° and 35°, who were treated with an underarm plastic brace, to 129 patients who were not treated. All patients were followed for 4 years. The patient’s thoracic or thoracolumbar curves were during this period evaluated for curve progression of more than 5°. Of the brace treated patients, 23 patients where lost to follow-up. Using survivorship analysis, a successful outcome was estimated in 74% of patients treated with a brace, compared to 34% of those who had no treatment; this difference was significant (P < 0.0001). In a retrospective study Lonstein and Winter [30] evaluated the result of brace treatment in a group of 1,020 patients over 35 years. From these patients a sub group of 177 had a curve between 20 and 29° and a Risser sign of 0–1. Using the natural history data of a similar group of non treated patients from their previous report [29] they where able to show in this sub-group a significant difference (P = 0.0001, chi-square test) of the failure rate between the group of treated patients and not treated patients. The not treated patients had a predicted failure rate of 68% and the failure rate of the braced patients in the sub group was 40%. We used the same report to predict the failure rate in our cohort. Although our results are promising, it is known that 5% of patients with a curve of less than 30° still progress to surgery during mature life [34]. We will follow our patients in whom the TriaC treatment effectively stopped curve progression before the surgical cut-off of 45° during mature life.
Besides affecting the lateral curve, most traditional braces reduce lordosis and kyphosis by tilting the pelvis. The purpose of pelvic tilt is to move the lumbar spine closer to the correction pads within the brace. According to Lindh et al. [28] reducing the lumbar lordosis may automatically lead to a reduction of the scoliosis as a result of a coupling mechanism between sagittal and lateral motions of the vertebra [38]. However, reduction of lumbar lordosis will also reduce thoracic kyphosis. The reduction of thoracic kyphosis is an unwanted effect, as reduced thoracic kyphosis already is an integral component of the scoliotic deformity [50]. Schaal et al. therefore, emphasise the need for a system that diminishes the effect of pelvic tilt on the thoracic kyphosis [43]. In the Triac brace the choice has been made to continuously apply corrective forces with the aim of reversing the deforming forces, without affecting the lordosis of the spine [37]. Growth is a continuous process and therefore, the correction forces should be applied continuously as well, even during the normal body motions of the patient.
In order to meet this requirement a flexible coupling, connecting the thoracic and lumbar parts of the brace is incorporated in the device. Most importantly, the forces exerted by the brace must be applied in such a way that they are maintained during all body motions of the patient. The new brace incorporates three separate functional elements: frame, springs and pelottes. The springs generate the orthotic forces, which are distributed by the frame and transmitted to the skin by the pelottes. The flexible coupling connects the thoracic and lumbar frame parts resulting in the application of constant forces that cannot be modified by the patients themselves (Fig. 7). The strength of the externally applied forces was chosen on the basis of literature data [10, 20, 27] Fig. 8.
Fig. 7TriaC-braceFig. 8Pre-brace Cobb-angle: 30° and 19° apical axial rotation. In the brace Cobb-angle: 17° and 9° apical axial rotation, sagittal curvature unchanged
The TriaC-brace provides a good primary correction of idiopathic scoliosis. There was a mean initial correction within the brace of 22 ± 26% for the primary curves and 28 ± 35% for the secondary curves, which is slightly less than reported in the literature [2, 30, 33, 34]. In the beginning of the study, we were not certain how patients would respond to the application of constant forces. Therefore, we started very carefully using relatively low forces. As we found that patients tolerated these forces well we subsequently increased the amount of force which resulted in improved initial corrections. According to the literature, achieved corrections gradually are lost following the termination of the brace treatment [7, 23, 30, 34, 49].
However, being efficacious is not all that is required of a brace; it should also be as comfortable as possible and cosmetically acceptable so that teenagers who require this treatment will actually use it. Non-acceptance of a brace by patients is a real and serious problem. Houghton et al. [24] placed a hidden transducer in their braces and found that actual compliance was considerably less than was reported by the patient; and only 20% of patients wore the brace as prescribed. Modern materials, lower profiles and reduced wearing times have all been tried to improve compliance and reduce the emotional difficulties experienced with brace wear. According to some authors, there is little difference in effect between part-time (12–16 h) and full-time (23 h) wearing of a brace [13, 21]. Kahanowitz [25] reported such findings but only if the pre-brace Cobb-angle were less than 35°; if larger than this more than 50% of curves would progress to such an extent that surgery became necessary.
The current study demonstrates that the TriaC brace reduces the scoliosis, and the achieved correction is maintained during brace treatment. It prevents further progression of the Cobb angle in idiopathic scoliosis. The new brace does not differ from the conventional braces as far as maintaining the deformity in the coronal plane is concerned. This is expected as the force system of the TriaC brace in the frontal plane is in accordance with the force system in the conventional braces.
The new brace offers more comfort to the patient and a better cosmetic appearance. With TriaC brace there are no restrictions regarding daily or sporting activities, and it can be worn with all types of clothing.
Summary
Compared with published studies on the natural history of idiopathic scoliosis, use of the Triac brace appeared to significantly improve the course of curves between 20 and 40° in skeletally immature individuals. Control or net correction of idiopathic scoliotic curves was achieved in 76% of patients.
The new dynamic brace offers more comfort to the patient and a better cosmetic appearance.

scoliosis
bracing
natural history
biomechanics