Morphological difference of Escherichia coli non-heme ferritin iron cores reconstituted in the presence and absence of inorganic phosphate.

The iron core of Escherichia coli ferritin was reconstituted in the presence and absence of phosphate. The core formed in the presence of phosphate contained phosphate in amounts comparable to the iron content. The size distribution of the core was analyzed by analytical ultracentrifugation. A continuous size distribution was observed in the presence of phosphate, whereas a multimodal distribution was found in the absence of phosphate. In the presence of phosphate, the core size observed by electron microscopy was consistent with the inner diameter of ferritin. In contrast to this, clusters of several smaller particles were observed in the absence of phosphate. The small-angle X-ray scattering was measured under contrast matching conditions to obtain information on the iron core shape. A fringe was observed in the scattering profile in the presence of phosphate, but it was not observed in the absence of phosphate. Combining all results, we conclude that a hollow spherical core was formed in the presence of phosphate, while several small particles were formed within the inner cavity in the absence of phosphate.

Structure, Function, Folding, and Aggregation of a Neuroferritinopathy-Related Ferritin Variant.

Neuroferritinopathy is a rare, adult-onset, dominantly inherited movement disorder caused by mutations in the ferritin gene. A ferritin light-chain variant related to neuroferritinopathy, in which alanine 96 is replaced with threonine (A96T), was expressed in Escherichia coli, purified, and characterized. The circular dichroism, analytical ultracentrifugation, and small-angle X-ray scattering studies have shown that both the subunit structure and the assembly of A96T are the same as those of wild-type human ferritin light chain (HuFTL). The iron-incorporation ability was also comparable to that of HuFTL. Although the structural stability against heat, acid, and denaturant was reduced, the structure was sufficiently stable under physiological conditions. The most remarkable defects observed for A96T were a lower refolding efficiency and a stronger propensity to aggregate. The possible relationship between folding deficiency and disease is discussed.

Predicting acetabular growth in developmental dysplasia of the hip following open reduction after walking age.

BACKGROUND: Acetabular dysplasia of the hip following open reduction can complicate the treatment of developmental dysplasia of the hip (DDH). The purposes of this retrospective study were to investigate the long-term results of open reduction performed via an extensive anterolateral approach for DDH after walking age and to predict acetabular development using postoperative radiographs and arthrograms. METHODS: From 1973 to 2001, we performed open reduction for 131 hips in 119 pediatric patients with DDH after failed closed reduction. Of these, 85 hips of 73 patients who underwent arthrography at 5 years old were followed-up radiologically until skeletal maturity. Mean age at the time of surgery was 17 ± 4.6 months (range, 10-33 months), and mean age at final survey was 19 ± 5.7 years (range, 14-33 years). Mean follow-up time was 17.7 ± 5.8 years (range, 13-32 years). Groups with satisfactory outcomes (66 hips) and unsatisfactory outcomes (19 hips) according to the Severin classification were compared. Factors predicting acetabular development were identified using univariate and multiple logistic analyses. RESULTS: Univariate analysis showed a significant between-group difference in acetabular index (AI) at 2 months postoperatively, and in center-edge (CE) angle, cartilaginous AI (CAI), and cartilaginous CE angle at 5 years old (p < 0.05 each). In multiple logistic regression analysis, CAI at 5 years old represented a predictor of acetabular development after open reduction for DDH (odds ratio, 1.81; 95% confidence interval (CI), 1.04-3.13; p < 0.05). Area under the receiver operating characteristic curve for CAI at 5 years old was 0.93 (95%CI, 0.85-1.0), and the optimal cut-off was 10° (81.8% sensitivity, 92% specificity). CONCLUSIONS: A CAI ≥10° on hip arthrograms at 5 years old may offer a useful indicator of the need for corrective surgery following open reduction after walking age.

ß-Strand twisting/bending in soluble and transmembrane ß-barrel structures.

The majority of ß-strands in globular proteins have a right-handed twist and bend. The dominant driving force for ß-strand twisting is thought to be inter-strand hydrogen bonds. We previously demonstrated that for water-soluble proteins, both the twisting and bending of ß-strand are suppressed by the polar side chains of serine, threonine, and asparagine residues. To determine whether this also holds for transmembrane ß-strands, we calculated and statistically analyzed the twist and bend angles of four-residue frames of ß-strands in both transmembrane and water-soluble ß-barrel proteins with known three-dimensional structures. In the case of transmembrane ß-strands, we found that twisting was suppressed even for frames not containing serine, threonine, or asparagine residues. The suppression of twisting in transmembrane ß-strands could be attributed to the propagation of the suppressive effect of serine, threonine, and asparagine residues within a frame to the neighboring, hydrogen-bonded strands under the restriction that all strands in the closed barrel structure must have similar twist angles. A similar tendency was also observed for water-soluble ß-barrel proteins. We previously showed that the dominant driving force for ß-strand bending is hydrophobic interactions involving aromatic residues within and outside the strand. Transmembrane ß-barrels have no hydrophobic core; however, rather hydrophilic residues predominate inside the barrel or the ß-strands of transmembrane ß-barrels have larger bend angles than those of water-soluble ß-barrels. Our results reveal that, in transmembrane ß-barrel proteins, the glycine-aromatic ring motif is important for generating the ß-strand bending necessary for barrel formation.

Optimization of Haemophilus influenzae adhesin transmembrane domain expression in Escherichia coli.

To obtain a high yield of the transmembrane domain of Haemophilus influenzae adhesin (HiaTD) in Escherichia coli, we attempted to express the HiaTD with and without a signal sequence using a T7 expression system. The expression level of HiaTD after induction was followed by quantification of the purified HiaTD, flow cytometric analysis of the outer membrane integrated HiaTD, and immunoblotting assay of fractionated cell lysate. In the expression system with a signal sequence, although the amount of cell-surface-expressed HiaTD increased over time, the number of HiaTD-expressing cells decreased, probably because of plasmid instability. As a result, the amount of purified HiaTD reached a plateau at 2 h postinduction. Although expression without the signal sequence provides a large amount of proteins as inclusion bodies in some membrane proteins, HiaTD expressed without a signal sequence was not observed as inclusion bodies and seemed to be assembled into the outer membrane during or after cell lysis.

Electrostatic Repulsion between Unique Arginine Residues Is Essential for the Efficient in Vitro Assembly of the Transmembrane Domain of a Trimeric Autotransporter.

Haemophilus influenzae adhesin (Hia) belongs to the trimeric autotransporter family and mediates the adherence of these bacteria to the epithelial cells of host organisms. Hia contains a passenger and a transmembrane domain. The transmembrane domain forms a 12-stranded ß-barrel in which four strands are provided by each subunit. The ß-barrel has a pore that is traversed by three α-helices. This domain has a unique arginine cluster, in which the side chains of the three arginine residues located at position 1077 (Arg1077) protrude into the pore of the ß-barrel. This arrangement seems to be unfavorable for assembly, because of repulsion between the positive charges. In this study, we investigated the in vitro assembly of the Hia transmembrane minimum domain (mHiaTD) and found that the dissociated mHiaTD reassembled in detergent solution. To investigate the role of Arg1077 in trimer assembly, we generated mutant proteins in which Arg1077 was replaced with methionine or lysine. The reassembly kinetics of the mutants was compared with that of the wild-type protein. The methionine mutant showed misassembly, whereas the lysine mutant showed reversible assembly, similar to that observed for the wild-type protein. These results show that electrostatic repulsion between the positive charges of Arg1077 is important for preventing the formation of misassembled oligomers by the mHiaTD in vitro.

An Analysis of the Characteristics and Improved Use of Newly Developed CT-based Navigation System in Total Hip Arthroplasty.

We developed a surface matching-type computed tomography (CT)-based navigation system for total hip arthroplasty (the N-navi; TEIJIN NAKASHIMA MEDICAL, Okayama, Japan). In the registration step, surface matching was performed with digitizing points on the pelvic bone surface after coarse paired matching. In the present study, we made model bones from the CT data of patients whose acetabular shapes had various deformities. We measured the distances and angles after surface matching from the fiducial points and evaluated the ability to correct surface-matching registration on each pelvic form, using several areas and numbers of points. When the surface-matching points were taken on the superior area of the acetabulum, the correction was easy for the external direction, but it was difficult to correct for the anterior and proximal directions. The correction was difficult for external and proximal directions on the posterior area. Each area of surface-matching points has particular directions that are easily corrected and other directions that are difficult to correct. The shape of the pelvis also affected the correction ability. Our present findings suggest that checking the position after coarse paired matching and choosing the surface-matching area and points that are optimal to correct will improve the accuracy of total hip arthroplasty and reduce surgical times.

Total hip arthroplasty after failed treatment of proximal femur fracture.

INTRODUCTION: Total hip arthroplasty (THA) is a good option as a salvage procedure after failed treatment of proximal femur fracture. The anatomy of the proximal femur, however, makes this surgery complicated and challenging. The purpose of this study was to evaluate the radiographic and clinical outcomes of THA after failed treatment of proximal femur fractures. MATERIALS AND METHODS: We retrospectively analysed 50 consecutive THAs (42 women, 8 men; mean age 77 years) after failed treatment of a proximal femur fracture. Mean postoperative follow-up was 58.1 months. Preoperative diagnoses were femoral neck fracture in 18 hips and trochanteric fracture in 32 hips, including three that were infected. Failure resulted from cutout in 22 cases, osteonecrosis in 12, non-union with failed fixation in nine, postoperative osteoarthritis in four, and infection in three. Factors compared included radiographic assessment, complication rate, visual analogue scale (VAS), and Harris Hip Scores (HHS). Radiographic variables included femoral neck anteversion and cup and stem alignment. RESULTS: Absolute values of the differences in femoral neck anteversion between the affected and healthy sides were 6.0° in the femoral neck fracture group and 19.2° in the trochanteric fracture group (p = 0.01). There were no significant differences in cup anteversion (p = 0.20) or stem anteversion (p = 0.08). The complication rate was significantly higher in the trochanteric fracture group than in the femoral neck fracture group (25 vs 0%, p < 0.0001). Postoperative complications in the trochanteric fracture group included three periprosthetic fractures (9.4%), two dislocations (6.3%), two surgical-site infections (6.3%), and one stem penetration (3.1%). Although no significant differences between groups were seen in the VAS or HHS at final follow-up (p = 0.32, 0.09, respectively), these measures were significantly improved at final follow-up in both groups (p < 0.0001 for both). CONCLUSIONS: Performing THA after failed treatment of trochanteric fractures requires consideration of complication risk and incorrect femoral neck anteversion.

Electrostatic Repulsion during Ferritin Assembly and Its Screening by Ions.

Escherichia coli non-heme-binding ferritin A (EcFtnA) is a spherical cagelike protein that is composed of 24 identical subunits. EcFtnA dissociates into 2-mers under acidic conditions and can reassemble into the native structure when the pH is increased. To understand how electrostatic interactions influence the assembly reaction, the dependence of the process on ionic strength and pH was investigated. The assembly reaction was initiated by stopped-flow mixing of the acid-dissociated EcFtnA solution and high-pH buffer solutions and monitored by time-resolved small-angle X-ray scattering. The rate of assembly increased with increasing ionic strength and decreased with increasing pH from 6 to 8. These dependences were thought to originate from repulsion between assembly units (2-mer in the case of EcFtnA) with the same net charge sign; therefore, to test this assumption, mutants with different net charges (net-charge mutants) were prepared. In buffers with a low ionic strength, the rate of assembly increased with a decreasing net charge. Thus, repulsion between the assembly unit net charges was demonstrated to be an important factor determining the rate of assembly. However, the difference in the assembly rate among net-charge mutants was not significant in buffers with an ionic strength of >0.1. Notably, under such high-ionic strength conditions, the assembly rate increased with an increasing ionic strength, suggesting that local electrostatic interactions are also responsible for the ionic strength dependence of the rate of assembly and are repulsive on average.

Ferritin Assembly Revisited: A Time-Resolved Small-Angle X-ray Scattering Study.

The assembly reaction of Escherichia coli ferritin A (EcFtnA) was studied using time-resolved small-angle X-ray scattering (TR-SAXS). EcFtnA forms a cagelike structure that consists of 24 identical subunits and dissociates into dimers at acidic pH. The dimer maintains nativelike secondary and tertiary structures and is able to reassemble into a 24-mer when the pH is increased. The reassembly reaction was induced by pH jump, and reassembly was followed by TR-SAXS. Time-dependent changes in the forward scattering intensity and in the gyration radius suggested the existence of a significant population of intermediate oligomers during the assembly reaction. The initial reaction was a mixture of second- and third-order reactions (formation of tetramers and hexamers) from the protein concentration dependence of the initial velocity. The time-dependent change in the SAXS profile was roughly explained by a simple model in which only tetramers, hexamers, and dodecamers were considered as intermediates.

A Physicochemical and Mutational Analysis of Intersubunit Interactions of Escherichia coli Ferritin A.

Ferritin A from Escherichia coli (EcFtnA) is 24-meric protein, which forms spherical cagelike structures called nanocages. The nanocage structure is stabilized by the interface around 4-, 3-, and 2-fold symmetric axes. The subunit structure of EcFtnA comprises a four-helix bundle (helices A-D) and an additional helix E, which forms a 4-fold axis. In this study, we examined the contribution of the interface around three symmetric axes. pH-induced dissociation experiments monitored by analytical ultracentrifugation and small-angle X-ray scattering showed that the dimer related by 2-fold symmetry is the most stable unit. Mutations located near the 3-fold axis revealed that the contribution of each interaction was small. A mutant lacking helix E at the 4-fold axis formed a nanocage, suggesting that helix E is not essential for nanocage formation. Further truncation of the C-terminus of helix D abrogated the formation of the nanocage, suggesting that a few residues located at the C-terminus of helix D are critical for this process. These properties are similar to those known for mammalian ferritins and seem to be common principles for nanocage formation. The difference between EcFtnA and mammalian ferritins was that helix E-truncated EcFtnA maintained an iron-incorporating ability, whereas mammalian mutants lost it.

The origin of ß-strand bending in globular proteins.

BACKGROUND: Many ß-strands are not flat but bend and/or twist. However, although almost all ß-strands have a twist, not all have a bend, suggesting that the underlying force(s) driving ß-strand bending is distinct from that for the twist. We, therefore, investigated the physical origin(s) of ß-strand bends. METHODS: We calculated rotation, twist and bend angles for a four-residue short frame. Fixed-length fragments consisting of six residues found in three consecutive short frames were used to evaluate the twist and bend angles of full-length ß-strands. RESULTS: We calculated and statistically analyzed the twist and bend angles of ß-strands found in globular proteins with known three-dimensional structures. The results show that full-length ß-strand bend angles are related to the nearby aromatic residue content, whereas local bend angles are related to the nearby aliphatic residue content. Furthermore, it appears that ß-strands bend to maximize their hydrophobic contacts with an abutting hydrophobic surface or to form a hydrophobic side-chain cluster when an abutting hydrophobic surface is absent. CONCLUSIONS: We conclude that the dominant driving force for full-length ß-strand bends is the hydrophobic interaction involving aromatic residues, whereas that for local ß-strand bends is the hydrophobic interaction involving aliphatic residues.

Venous thromboembolism after total hip arthroplasty diagnosed by enhanced computed tomography:comparison of selective thromboprophylaxis and no thromboprophylaxis.

Total hip arthroplasty (THA) is the most effective treatment for advanced or end-stage hip osteoarthritis. However, venous thromboembolism (VTE) remains one of its unresolved complications. We reviewed the records of 322 patients undergoing primary THA and investigated the efficacy of anticoagulant prophylaxis for VTE. Our study cohort consisted of 60 patients who received no anticoagulants, 100 patients who received a factor Xa inhibitor (fondaparinux), 100 patients who received low molecular weight heparin (enoxaparin), and 62 patients who selectively received no anticoagulant prophylaxis due to perioperative bleeding, weight, and/or hemoglobin concentration. Enhanced 64-slice multidetector row computed tomography was performed postoperatively for 7 days in all cases. The incidence of VTE in the four groups was 15%, 9.0%, 6.0%, and 6.4%, respectively. The incidence of VTE was significantly lower in the groups receiving anticoagulant prophylaxis and the group selectively receiving no anticoagulant prophylaxis than in the group receiving no anticoagulants. Complications of fondaparinux therapy included hepatic dysfunction in 4 cases (4.0%), minor bleeding in 2 cases (2.0%), persistent wound drainage in 3 cases (3.0%), and eruption in 1 case (1.0%). The complications of enoxaparin therapy were persistent wound drainage in 1 case (1.0%) and progression of anemia in 1 case (1.0%). The incidence of VTE was low in patients who selectively received no anticoagulant prophylaxis, so we conclude that anticoagulant prophylaxis should be used selectively in THA cases.

Erratum. Venous Thromboembolism after Total Hip Arthroplasty Diagnosed by Enhanced Computed Tomography:Comparison of Selective Thromboprophylaxis and No Thromboprophylaxis.

Total hip arthroplasty (THA) is the most effective treatment for advanced or end-stage hip osteoarthritis. However, venous thromboembolism (VTE) remains one of its unresolved complications. We reviewed the records of 322 patients undergoing primary THA and investigated the efficacy of anticoagulant prophylaxis for VTE. Our study cohort consisted of 60 patients who received no anticoagulants, 100 patients who received a factor Xa inhibitor (fondaparinux), 100 patients who received low molecular weight heparin (enoxaparin), and 62 patients who selectively received no anticoagulant prophylaxis due to perioperative bleeding, weight, and/or hemoglobin concentration. Enhanced 64-slice multidetector row computed tomography was performed postoperatively for 7 days in all cases. The incidence of VTE in the four groups was 15%, 9.0%, 6.0%, and 6.4%, respectively. The incidence of VTE was significantly lower in the groups receiving anticoagulant prophylaxis and the group selectively receiving no anticoagulant prophylaxis than in the group receiving no anticoagulants. Complications of fondaparinux therapy included hepatic dysfunction in 4 cases (4.0%), minor bleeding in 2 cases (2.0%), persistent wound drainage in 3 cases (3.0%), and eruption in 1 case (1.0%). The complications of enoxaparin therapy were persistent wound drainage in 1 case (1.0%) and progression of anemia in 1 case (1.0%). The incidence of VTE was low in patients who selectively received no anticoagulant prophylaxis, so we conclude that anticoagulant prophylaxis should be used selectively in THA cases.

Delineation of solution burst-phase protein folding events by encapsulating the proteins in silica gels.

Many studies have shown that during the early stages of the folding of a protein, chain collapse and secondary structure formation lead to a partially folded intermediate. Thus, direct observation of these early folding events is crucial if we are to understand protein-folding mechanisms. Notably, these events usually manifest as the initial unresolvable signals, denoted the burst phase, when monitored during conventional mixing experiments. However, folding events can be substantially slowed by first trapping a protein within a silica gel with a large water content, in which the trapped native state retains its solution conformation. In this study, we monitored the early folding events involving secondary structure formation of five globular proteins, horse heart cytochrome c, equine ß-lactoglobulin, human tear lipocalin, bovine α-lactalbumin, and hen egg lysozyme, in silica gels containing 80% (w/w) water by CD spectroscopy. The folding rates decreased for each of the proteins, which allowed for direct observation of the initial folding transitions, equivalent to the solution burst phase. The formation of each initial intermediate state exhibited single exponential kinetics and Arrhenius activation energies of 14-31 kJ/mol.

Local sequence of protein ß-strands influences twist and bend angles.

ß-Sheet twisting is thought to be mainly determined by interstrand hydrogen bonds with little contribution from side chains, but some proteins have large, flat ß-sheets, suggesting that side chains influence ß-structures. We therefore investigated the relationship between amino acid composition and twists or bends of ß-strands. We calculated and statistically analyzed the twist and bend angles of short frames of ß-strands in known protein structures. The most frequent twist angles were strongly negatively correlated with the proportion of hydrophilic amino acid residues. The majority of hydrophilic residues (except serine and threonine) were found in the edge regions of ß-strands, suggesting that the side chains of these residues likely do not affect ß-strand structure. In contrast, the majority of serine, threonine, and asparagine side-chains in ß-strands made contacts with a nitrogen atom of the main chain, suggesting that these residues suppress ß-strand twisting.

Relationship between chain collapse and secondary structure formation in a partially folded protein.

Chain collapse and secondary structure formation are frequently observed during the early stages of protein folding. Is the chain collapse brought about by interactions between secondary structure units or is it due to polymer behavior in a poor solvent (coil-globule transition)? To answer this question, we measured small-angle X-ray scattering for a series of ß-lactoglobulin mutants under conditions in which they assume a partially folded state analogous to the folding intermediates. Mutants that were designed to disrupt the secondary structure units showed the gyration radii similar to that of the wild type protein, indicating that chain collapse is due to coil-globule transitions.

Individual Bilateral Difference of Femur, Tibia, and Leg Rotation: A Clinical Study of 141 Healthy Japanese Individuals Using Computed Tomography.

Background The malrotation of a femur and tibial fracture after surgery has been described in many articles. However, these studies have not considered individual bilateral differences (IBDs). The IBD of femur and tibial rotation has been identified via computed tomography (CT) in recent American studies. The IBD in rotation should be considered during femur and tibial surgery. However, IBDs in femur and tibial rotation remain unknown in the Japanese population. This study aimed to evaluate the rotation of the femur, knee, tibia, and leg, sex differences, and IBD in rotation among Japanese individuals with healthy bones by using CT analysis. Materials and methods In total,141 patients who underwent CT angiography or venography were included (70 men, 71 women; mean age, 44.7 years). The bilateral axial femur, knee, tibia, and leg rotation alignment were independently measured. The distribution, sex, and IBD were analyzed. The IBD in rotation had two statistical factors: absolute bilateral difference (ABD) and relative bilateral difference (RBD). Results The mean ABD of femur rotation was 6.5°, and the distribution of ABD of femur rotation ≤15° was 95%. The mean ABD of tibia rotation was 5.1°, and the distribution of ABD of tibia rotation ≤10° was 89%. The RBD of femur rotation was not significantly different between the right and left sides. The RBD of tibia rotation showed a higher mean external rotation of 3.3° on the right side (<0.001). The Pearson correlation coefficients of the femur, knee, tibia, and leg rotation between the right and left sides were high (r= 0.702-0.81; all, p<0.001). All elements of rotation showed significant differences between men and women, whereas the ABD and RBD of all elements showed no significant difference. Conclusion The distributions of ABD in femur and tibia rotation supported the previous definition of an acceptable rotation difference between the normal and fractured femur and tibia of ≤15°and ≤10°, respectively. The possibility of higher external rotation on the right side needs to be taken into account during tibial surgery.

Time elapsed from definitive diagnosis to surgery for osteonecrosis of the femoral head: a nationwide observational study in Japan.

OBJECTIVES: This study documents the time elapsed from the diagnosis of osteonecrosis of the femoral head (ONFH) to surgery, exploring the factors that influence ONFH severity. DESIGN: Retrospective observational study of a nationwide database. SETTING: The Kaplan-Meier method with log-rank tests was applied to examine the period from definitive diagnosis of ONFH to surgery using any surgery as the end point. For bilateral cases, the date of the first surgery was the endpoint. PARTICIPANTS: This study included 2074 ONFH cases registered in 34 university hospitals and highly specialised hospitals of the multicentre sentinel monitoring system of the Japanese Investigation Committee between 1997 and 2018. MAIN OUTCOME MEASURE: The primary outcome was the time from diagnosis to surgery. The secondary outcome was the proportion of subjects remaining without surgery at 3, 6 and 9 months, and at 1, 2 and 5 years after diagnosis. RESULTS: The median time to surgery was 9 months (IQR 4-22 months) after diagnosis of ONFH. The time to surgery was significantly shorter in the alcohol alone group and the combined corticosteroid and alcohol group than in the corticosteroid alone group (p=0.018 and p<0.001, respectively), in early stage ONFH with no or mild joint destruction (stages II and III, p<0.001), and with joint preserving surgery (p<0.001). The proportion without surgery was 75.8% at 3 months, 59.6% at 6 months, 48.2% at 9 months, 40.5% at 1 year, 22.2% at 2 years and 8.3% at 5 years. CONCLUSION: ONFH has been considered to be an intractable disease that often requires surgical treatment, but the fact that surgery was performed in more than half of the patients within 9 months from diagnosis suggests severe disease with a significant clinical impact. TRIAL REGISTRATION NUMBER: Chiba University ID1049.

A new navigation system for minimally invasive total knee arthroplasty.

A computer-assisted navigation system to be used for total knee arthroplasties (TKAs) was reported to improve the accuracy of bone resection and result in precise implant placement, but the concomitant surgical invasion and time consumption are clinical problems. We developed a computed tomography (CT)-based navigation system (NNS) to be used for minimally invasive TKA. It requires only the reference points from a small limited area of the medial femoral condyle and proximal tibia through a skin incision to obtain optical images. Here we evaluated the usefulness and accuracy of the NNS in comparison with the commercially available BrainLAB image-free navigation system (BLS). In a clinical experiment, the registration times obtained with the NNS tended to be shorter than those obtained with the BLS, but not significantly so. The NNS group tended to be in the extended position in the sagittal plane of the distal femur within 3 degrees, and the BLS group showed rather flexed deviation in the sagittal plane of the anterior femur.