Sperm mRNA screening by reverse transcription-recombinase polymerase amplification (RT-RPA) assay: Decision-making for differential extraction.

The presence of sperm cells is an indicator for differential extraction on sexual assault samples. In general, sperm cells are identified by microscopic analysis; however, this conventional method takes time and effort, even for trained personnel. Here, we present a reverse transcription-recombinase polymerase amplification (RT-RPA) assay targeting sperm mRNA marker (PRM1). The RT-RPA assay requires only 40 min for PRM1 detection and demonstrates a sensitivity of 0.1 µL of semen. Our results indicate that the RT-RPA assay may be a rapid, simple, and specific strategy for screening sperm cells in sexual assault samples.

Loop-mediated isothermal amplification assay for fluorescence analysis and lateral flow detection of male DNA.

Male DNA screening is important in forensic investigations, such as sexual assault cases. Although quantitative real-time PCR is a robust method for detection of male DNA, it is time-consuming and labor-intensive. We herein report the development of a male DNA-targeted loop-mediated isothermal amplification (LAMP) assay that can be used for both laboratory-based fluorescence analysis and on-site lateral flow detection. The two detection systems are independent, but we streamlined the reaction before the detection by introducing a fluorescence probe and biotin-labeled primer into a single reaction. This allowed the evaluation of fluorescence signal followed by lateral flow detection. Both the fluorescence and lateral flow analyses detected as low as 10 pg of male DNA. We also integrated an alkaline lysis method with our LAMP assay. The direct assay successfully detected male DNA from forensic samples without purification. The workflow requires only <40 min for fluorescence analysis and <45 min for lateral flow detection. Furthermore, when combined with a lateral flow strip, this workflow does not require any sophisticated instruments. These findings suggest that our assay is a promising strategy for on-site male DNA screening as well as laboratory-based testing.

Rapid screening method for male DNA by using the loop-mediated isothermal amplification assay.

Screening for male-derived biological material from collected samples plays an important role in criminal investigations, especially those involving sexual assaults. We have developed a loop-mediated isothermal amplification (LAMP) assay targeting multi-repeat sequences of the Y chromosome for detecting male DNA. Successful amplification occurred with 0.5 ng of male DNA under isothermal conditions of 61 to 67 °C, but no amplification occurred with up to 10 ng of female DNA. Under the optimized conditions, the LAMP reaction initiated amplification within 10 min and amplified for 20 min. The LAMP reaction was sensitive at levels as low as 1-pg male DNA, and a quantitative LAMP assay could be developed because of the strong correlation between the reaction time and the amount of template DNA in the range of 10 pg to 10 ng. Furthermore, to apply the LAMP assay to on-site screening for male-derived samples, we evaluated a protocol using a simple DNA extraction method and a colorimetric intercalating dye that allows detection of the LAMP reaction by evaluating the change in color of the solution. Using this protocol, samples of male-derived blood and saliva stains were processed in approximately 30 min from DNA extraction to detection. Because our protocol does not require much hands-on time or special equipment, this LAMP assay promises to become a rapid and simple screening method for male-derived samples in forensic investigations.

Simulation of the optimal femoral insertion site in medial patellofemoral ligament reconstruction.

PURPOSE: To develop and validate a new simulation system to predict the optimal femoral insertion site of the medial patellofemoral ligament (MPFL) using preprogrammed conditions of graft length change. METHODS: A computed tomography scan was performed for constructing bone surface models of the patella and the femur. Lateral radiographs of the knee and axial radiographs of the patellofemoral joint at knee flexion were used for constructing a three-dimensional patellofemoral joint model by 3D-2D image matching at each knee flexion angle. To determine the optimal femoral insertion site, the following three conditions were devised: (1) MPFL behaves isometric from 0° to 60°, (2) MPFL is most taut at full extension, and (3) MPFL is slack at more than 60° of knee flexion. Every condition was applied to the femoral bone surface model, and the area that fulfilled all three conditions was defined as the optimal femoral insertion site of MPFL. Twenty knees of ten healthy volunteers were assessed to verify the simulation. Comparison between simulated optimal femoral insertion and anatomical insertion was done. RESULTS: The mean simulated optimal femoral insertion was close to the anatomical insertion site. There were no significant differences in the proximal-distal position and anterior-posterior position between the simulated insertion and the anatomical insertion sites. CONCLUSION: The system can be useful for predicting the optimal femoral insertion site as a part of a preoperative plan for MPFL reconstruction, and it may help surgeons to determine the optimal femoral insertion site during MPFL reconstruction.

The influence of the tibial slope on intra-operative soft tissue balance in cruciate-retaining and posterior-stabilized total knee arthroplasty.

PURPOSE: This study aims to make clear the influence of the tibial slope on intra-operative soft tissue balance measurements using a tensor in cruciate-retaining and posterior-stabilized total knee arthroplasty (TKA). METHODS: Forty patients with osteoarthritis of the knee received TKAs (20 cruciate-retaining TKAs and 20 posterior-stabilized TKA). Soft tissue balance was measured using an offset type tensor at 0, 10, 45, 90, 135 degrees of knee flexion. The tibial slopes were measured by post-operative lateral radiograph. The correlation between the tibial slope and values of soft tissue balance were assessed. RESULTS: Joint component gap at 90° (R = 0.537, p < 0.01) and 135° (R = 0.463, p < 0.05) of flexion and joint component gap change value of 90-0° (R = 0.433, p < 0.05) showed positive correlations with tibial slope in posterior-stabilized TKA. There was no relationship between the tibial slope and the value of soft tissue balances in cruciate-retaining TKA. CONCLUSIONS: In the present study, we confirmed that increasing the tibial slope resulted in a larger flexion gap compared to extension gap in posterior-stabilized TKA. Surgeons should be aware that increasing the tibial slope is one factor responsible for widening the flexion-extension gap difference in posterior-stabilized TKA.

No difference between double-high insert and medial-pivot insert in TKA.

PURPOSE: To compare the clinical midterm results in ADVANCE total knee arthroplasty (TKA) with double-high (DH) insert, with same type implant with medial-pivot (MP) insert. METHOD: Forty ADVANCE TKAs were randomly divided into two groups, and two different design insert, DH insert, and MP insert were used in each group. At midterm, 4-5 years after surgery, Knee Society Scores (KSS), Knee Society Functional Scores (KSFS), range of motion (ROM), and UCLA activity score were assessed and reported in this study. RESULTS: Midterm clinical results, including ROM and KSS, were comparable with both groups. KSFS and UCLA activity score were equally good between the two groups. CONCLUSION: The results in this study revealed equally good clinical results with these types of implants at midterm follow-up, although the significant better ROM has not achieved by using DH insert. We concluded that the selection of inserts only could not achieve the better clinical results, including ROM and activity level in this study. LEVEL OF EVIDENCE: Therapeutic studies-investigating the results of treatment, Level II.

Tibial internal rotation is affected by lateral laxity in cruciate-retaining total knee arthroplasty: an intraoperative kinematic study using a navigation system and offset-type tensor.

PURPOSE: The purpose of this study was to test the hypothesis that intraoperative soft-tissue balance assessed by an offset-type tensor influences post-operative knee kinematics after cruciate-retaining (CR) total knee arthroplasty (TKA). METHODS: The influence of intraoperative soft-tissue balance on knee kinematics in CR-TKA was retrospectively analysed in 30 patients. Intraoperative soft-tissue balance parameters such as varus angle (varus ligament balance), joint component gap (centre gap), and medial and lateral compartment gaps were measured in the navigation system while applying 40-lb joint distraction force at 0°, 10°, 30°, 60°, 90°, and 120° of knee flexion using an offset-type tensor with the patella reduced. Tibial internal rotation and tibial anterior translation were measured as the differences between the values at 60° and 120° of flexion using the navigation system. Correlations between the soft-tissue parameters and post-operative knee kinematics were analysed. RESULTS: The varus ligament balance was positively correlated with tibial internal rotation at 60° and 90° of flexion (R = 0.54, P < 0.05; R = 0.60, P < 0.01, respectively). Furthermore, the joint component gap was positively correlated with tibial internal rotation at 90° of flexion (R = 0.44, P < 0.05), and the lateral compartment gap was positively correlated with tibial internal rotation at 60°, 90°, and 120° of knee flexion. CONCLUSIONS: The intraoperative varus ligament balance and joint component gap values were factors that predicted post-operative knee kinematics after CR-TKA. Lateral laxity at mid-to-deep knee flexion plays a significant role in tibial internal rotation. LEVEL OF EVIDENCE: III.

Female sample screening using colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) targeting non-coding RNA XIST.

Forensic sample screening is important for establishing an effective DNA typing workflow. The detection of sex-specific markers in forensic samples highlights the necessity for further analysis. Y-chromosome DNA can confirm male contributions, but female contributions are difficult to confirm using DNA-based methods. To address this, we developed a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the long non-coding RNA X-inactive specific transcript (XIST) to screen female samples. Operating at 65 °C for 30 min, the assay yielded results discernible from the color change of the pH indicator dye. The assay showed a detection limit of approximately 0.5 µL of blood. The assay also detected XIST RNA in mixed body fluids and mock samples, indicating its potential applicability to casework samples. Taken together, our assay provides a rapid and simple strategy for screening female samples.

RNA-based short tandem repeat analysis on the D18S51 locus.

DNA typing based on short tandem repeat (STR) analysis is an effective forensic method for human identification. Some STRs are contained within the introns of protein-coding genes and are transcribed as pre-mRNAs. However, the possibility of using RNA for STR analysis is yet to be fully explored. Considering that RNA in forensic samples is relatively stable, especially under dry- and low-temperature conditions, we hypothesized that STR information could be obtained from RNA. Here, we investigated the possibility of conducting RNA-based STR analysis using the D18S51 locus as a model.

Autophagy modulates osteoarthritis-related gene expression in human chondrocytes.

OBJECTIVE: Autophagy, an evolutionarily conserved process for the bulk degradation of cytoplasmic components, serves as a cell survival mechanism. The purpose of this study was to elucidate the role of autophagy in human chondrocytes and pathophysiology of osteoarthritis (OA). METHODS: Autophagy in articular cartilage and primary chondrocytes was assessed using antibodies for the autophagy markers light chain 3 and beclin 1. The states of autophagy under catabolic and nutritional stresses were examined. We also examined the effects of inhibition or induction of autophagy under stimulation with interleukin-1ß. Autophagy was inhibited by small interfering RNA targeting ATG5, and autophagy was induced by rapamycin. The effects of inhibition or induction of autophagy were examined by real-time polymerase chain reaction for aggrecan, COL2A1, MMP13, and ADAMTS5 messenger RNA. To further examine the mechanism of autophagy regulation in OA human chondrocytes, we investigated whether autophagy modulates apoptosis and reactive oxygen species (ROS). RESULTS: Autophagy was increased in OA chondrocytes and cartilage. Catabolic and nutritional stresses increased autophagy. In addition, the inhibition of autophagy caused OA-like gene expression changes, while the induction of autophagy prevented them. Furthermore, the inhibition of autophagy increased the amount of cleaved poly(ADP-ribose) polymerase and cleaved caspase 9, while the induction of autophagy inhibited these increases. ROS activity was also decreased by induction of autophagy. CONCLUSION: These observations suggested that increased autophagy is an adaptive response to protect cells from stresses, and that autophagy regulates OA-like gene expression changes through the modulation of apoptosis and ROS. Further studies about autophagy in chondrocytes will provide novel insights into the pathophysiology of OA.

Biomechanical analysis of the knee with partial anterior cruciate ligament disruption: quantitative evaluation using an electromagnetic measurement system.

PURPOSE: To investigate the biomechanical function of anterior cruciate ligament (ACL) remnants in ACL-deficient knees with both partial and complete tears. METHODS: Twenty partial ACL-deficient (group P), 20 complete ACL-deficient (group C), and 40 contralateral ACL-intact knees were examined. The end point during the Lachman test, side-to-side differences of KT-1000 measurements, and the pivot shift test were evaluated. Additionally, the side-to-side difference of anterior tibial translation during the Lachman test and the acceleration during the pivot shift test were calculated using an electromagnetic measurement system (EMS). RESULTS: The end point was found in 9 patients in group P, whereas it was not detected in group C. In KT-1000 measurements, the mean side-to-side differences were 3.8 ± 2.4 mm in group P and 5.4 ± 2.3 mm in group C. There was a significant difference between these 2 groups (P < .05). In the pivot shift test evaluation in group P, one patient was evaluated as grade 0, 17 patients as grade 1+, and 2 patients as grade 2+. In group C, 10 patients were evaluated as grade 1+, 9 patients as grade 2+, and one patient as grade 3+. Using the EMS, mean side-to-side differences during the Lachman test were 3.1 ± 2.1 mm in group P and 7.2 ± 3.2 mm in group C. The anterior-posterior displacement in group P was significantly less than that in group C (P < .05). In the quantitative pivot shift test, the mean acceleration in the contralateral ACL-intact knees was -632.7 ± 254.5 mm/s(2), whereas it was -1107.5 ± 398.9 mm/s(2) in group P and -1652.2 ± 754.9 mm/s(2) in group C. Significant differences were detected between the 3 knee conditions (P < .05). CONCLUSIONS: The quantitative assessments of knees with partial ACL ruptures during the Lachman test and the pivot shift test using the EMS showed less laxity than did knees with complete ACL tears, whereas their laxity was greater than the contralateral knees with intact ACLs. LEVEL OF EVIDENCE: Level III, diagnostic study of nonconsecutive patients.

Medial patellofemoral ligament reconstruction with lateral soft tissue release in adult patients with habitual patellar dislocation.

PURPOSE: Habitual dislocation of the patella is a rare condition and a treatment plan has not yet been established. The purpose of this case report is to assess the effectiveness of medial patellofemoral ligament (MPFL) reconstruction for treating adults with habitual dislocation. METHODS: We performed MFPL reconstruction in two women with habitual patellar dislocation. Patient 1 was 39 and Patient 2 was 57 years old. Both patients frequently experienced giving way and received surgical treatments that involved extensive release of tight lateral soft tissue followed by medial patellofemoral ligament reconstruction using semitendinosus tendon. RESULTS: Follow-ups at 5 years (Patient 1) and 3 years (Patient 2) after operation confirmed no recurrent dislocation in either patient and significantly improved knee function. CONCLUSIONS: Although a longer-term follow-up is necessary, MPFL reconstruction combined with lateral soft tissue release seems a promising treatment for habitual dislocation of the patella in adults. LEVEL OF EVIDENCE: V.

An analysis of surface profile for cylindrical osteochondral grafts of the knee quantitative evaluation using a three-dimensional laser scanner.

PURPOSE: To investigate the congruency of the articular cartilage surface of the knee between the recipient and donor site during autogenous osteochondral grafting using a three-dimensional (3D) laser scanning. METHODS: Six cadaveric knees were included in this study. The 3D profiles of the articular surface were obtained by a 3D laser scanner (FastSCAN(®), Polhemus). We divided each of the donor and recipient sites into 6 areas in each. The 2 central areas of the donor site were excluded from evaluation because of the trochlear groove. In the donor site, the peripheral and the middle one-third of the femoral articular surface in the medial and lateral patellofemoral joint were extracted. In the recipient site, the peripheral and the middle one-third of the articular surface in the medial and lateral femoral condyle were assessed. In each recipient area, vertical intervals (VIs) of grafts of 6, 8, and 10 mm diameter, showing the distance between highest and lowest point of articular surface were calculated from the data obtained and to the donor sites for matching. RESULTS: ϕ6- and ϕ8-mm grafts The VI of the middle area of the donor site did not differ significantly from that of either the peripheral or the middle area of the recipient site. The VI of the peripheral area of the donor site was significantly higher than that of the peripheral area of the recipient site (p < 0.01). ϕ10-mm grafts The VI of the middle area of the donor site was significantly lower than that of the peripheral area of the recipient site. The VI of the peripheral area of the donor site was significantly higher than that of the middle area of the recipient site (p < 0.01). CONCLUSIONS: An osteochondral graft harvested from the peripheral area of the patellofemoral joint might protrude into the middle area in the recipient site, whereas a ϕ10-mm osteochondral graft harvested from the middle area might be depressed from the peripheral area into the recipient site.

Different pattern in gap balancing between the cruciate-retaining and posterior-stabilized total knee arthroplasty.

PURPOSE: In order to permit soft tissue balance under more physiological conditions during total knee arthroplasties (TKAs), an offset-type tensor was developed to obtain soft tissue balancing throughout the range of motion with reduced patello-femoral (PF) and aligned tibiofemoral joints. The main purpose of the present study was to assess intra-operative soft tissue balance using a navigation system with the offset-type tensor in both cruciate-retaining (CR) and posterior-stabilized (PS) TKAs. METHODS: One hundred and twenty TKAs--80 CR and 40 PS--were performed in patients with varus-type osteoarthritis using a computed tomography-free navigation system. The offset-type TKA tensor with a reduced and repaired PF joint and femoral component in place was used with the tibia first gap technique to balance soft tissues (joint component gap and ligament balance) at 0°, 10°, 30°, 60°, 90°, and 120° of flexion. The achievement in equalized rectangular gap at extension and flexion--joint component gap within ±3 mm between extension and flexion and ligament balance within ±3° at extension and flexion--was assessed retrospectively. RESULTS: Both types of implants showed similar patterns of soft tissue balance throughout the range of motion, whereas PS TKA had larger values especially at 60° or 90° of flexion than did CR TKA. In the achievement of equalized rectangular gaps at extension and flexion, CR TKA was superior to PS TKA. CONCLUSION: Using the tibia first gap technique with the tensor allows appropriate soft tissue balancing, especially in CR TKA. LEVEL OF EVIDENCE: Therapeutic studies, Level II.

The influence of patella height on soft tissue balance in cruciate-retaining and posterior-stabilised total knee arthroplasty.

PURPOSE: Although the patella reduced or everted position has recently been recognised as an important factor influencing soft tissue balance during assessment in total knee arthroplasty (TKA), the influence of patella height on soft tissue balance has not been well addressed. Therefore, the relationship between soft tissue balance and patella height was investigated and differences between cruciate-retaining (CR) and posterior-stabilised (PS) TKA were compared. METHODS: Forty consecutive patients blinded to the type of implant received, were randomised prospectively. Using lateral radiographs, pre-operative patella height was measured. Using an offset-type tensor designed to measure the soft tissue balance with a reduced patellofemoral (PF) joint and femoral component in place, soft tissue balance was intra-operatively assessed in CR TKA (n = 20) and PS TKA (n = 20) in osteoarthritic patients. The joint component gap and varus ligament balance at zero, ten, 45, 90 and 135° of knee flexion with the patella reduced were measured. RESULTS: In PS TKA, the joint component gap positively correlated with patella height at 90 and 135° of knee flexion. However, there was no correlation between joint component gap and patella height at other flexion angles in PS TKA and any flexion angle in CR TKA. Varus ligament balance showed no significant correlation with patella height in either CR or PS TKA. CONCLUSION: Analysis of soft tissue balance and patella height only showed a positive correlation in joint component gap at a high flexion angle (90 and 135°) in PS TKA but not in other parameters examined. Pre-operative measurement of patella height may be an important factor for predicting an intra-operative flexion gap in PS TKA.

Kinematic factors affecting postoperative knee flexion after cruciate-retaining total knee arthroplasty.

PURPOSE: The purpose of this study was to investigate kinematic factors affecting postoperative knee flexion after cruciate-retaining (CR) total knee arthroplasty (TKA) by analysing pre- and postoperative knee kinematics. METHODS: We retrospectively analysed 58 patients with osteoarthritis who received the same implant series. Pre- and postoperative kinematics were measured intraoperatively using a navigation system. As a clinical outcome, we measured the knee flexion angle before and one year after surgery. Correlations among pre- and postoperative kinematics and postoperative flexion were analysed using simple linear regression analyses. RESULTS: Preoperative knee kinematics, including tibial internal rotation and anterior translation (R = 0.87, P < 0.001; R = 0.53, P < 0.001, respectively), were significantly correlated with postoperative kinematics. Preoperative varus-valgus movements improved significantly postoperatively; however, tibial internal rotation remained unchanged. Furthermore, postoperative knee flexion angle was significantly correlated with postoperative tibial internal rotation (R = 0.45, P < 0.001). CONCLUSIONS: Preoperative knee kinematics were unchanged even after CR-TKA. Postoperative tibial internal rotation is one of the most important factors affecting postoperative knee flexion.

Duplex loop-mediated isothermal amplification assay for simultaneous detection of human and human male DNA.

OBJECTIVE: Screening of human and human male DNA is necessary for forensic DNA analyses. Although quantitative real-time PCR (qPCR) is commonly used for detecting and quantifying these DNA targets, its use as a screening tool is time-consuming and labor-intensive. To streamline and simplify the screening process, we aimed to develop a duplex loop-mediated isothermal amplification (LAMP) assay capable of simultaneously detecting human and human male DNA in a single tube. We assessed the duplex LAMP assay for forensic application. RESULTS: For our duplex LAMP assay, we have utilized two fluorescent probes with HEX and FAM fluorophores to specifically detect human and human male DNA, respectively. The HEX (human target) signal was detected from both the male and female DNA samples, and the FAM (male target) signal was detected from only the male DNA sample. This assay has a sensitivity of 10-1 pg of DNA for both targets. Additionally, we successfully detected the two targets in the DNA samples extracted from forensically relevant body fluids, including blood, saliva, semen, and vaginal secretions.

Improved reverse transcription-recombinase polymerase amplification assay for blood mRNA screening: comparison with one-step RT-qPCR assay.

mRNA profiling is effective for body fluid identification because of its sensitivity, specificity, and multiplexing capability. Body fluid mRNA markers can typically be detected using RT-qPCR, RT-PCR followed by capillary electrophoresis, or targeted RNA sequencing. However, due to the multiple handling steps involved, the analysis of many forensic samples using these methods requires time and effort. Here, we describe a rapid and simple method for detecting the blood mRNA marker hemoglobin ß (HBB), intended for use in screening before definitive blood identification. We employed a reverse transcription-recombinase polymerase amplification (RT-RPA) assay that can detect target mRNA within 20 min in a single tube. For comparison, we used a one-step RT-qPCR assay. We optimized the RT-RPA assay and found that it could detect HBB from 10-3-10-4 ng of leukocyte RNA and approximately 10-3 µL of blood. The sensitivity was 10-fold lower than that of the one-step RT-qPCR assay but higher than that of the comprehensive analysis methods for definitive blood identification. Thus, the rapidity and sensitivity of the RT-RPA assay support its use as a screening tool. We also found that the RT-RPA assay was highly tolerant to common inhibitors such as humic acid, hematin, tannic acid, and melanin. Considering the inhibitor tolerability, we integrated a simple lysis method (addition of TCEP/EDTA and heating at 95 °C for 5 min) without the RNA purification process into the RT-RPA assay. This direct assay successfully detected HBB in crude blood samples. Our findings suggest that the RT-RPA assay for HBB is a promising strategy for mRNA-based blood screening.

One-tube, two-step isothermal amplification of histatin 3 mRNA for saliva screening.

Saliva samples are frequently collected at crime scenes. Salivary mRNA profiling, such as that of histatin 3 (HTN3), is a highly specific approach that overcomes the limitation of traditional amylase tests. However, typical mRNA detection methods based on reverse transcription PCR (RT-PCR) are time-consuming and labor-intensive. Here, we report a one-tube, two-step isothermal amplification assay for HTN3 mRNA, which enables rapid, simple, and sensitive screening of saliva. The first step is an RT-recombinase polymerase amplification (RT-RPA) assay at 42 °C for 20 min; the second step is a loop-mediated isothermal amplification (LAMP) assay at 65 °C for 30 min. The reactions can be performed in a closed tube, and the products are detected using real-time fluorescence analysis. The assay sensitivity was 0.5 µL of saliva samples. It also detected HTN3 mRNA in mixed and mock samples, demonstrating its applicability to actual forensic samples. These findings suggest that our strategy is promising for screening of saliva from forensic samples.

Influence of intra-operative joint gaps on post-operative flexion angle in posterior cruciate-retaining total knee arthroplasty.

PURPOSE: The purpose of this study is to examine the joint component gap and its relationship with post-operative flexion angles in posterior cruciate-retaining (CR) total knee arthroplasty (TKA). In posterior-stabilized (PS) TKA, an inverse correlation between the joint component gap and the post-operative flexion angle was reported. However, the kinematics of the joint component gap has a different pattern in PS and CR TKA. It was hypothesised that CR TKA has a different correlation between the joint component gap and the post-operative flexion angle compared to PS TKA. METHODS: The joint component gap was measured with an offset-type tensor. The joint component gaps were measured at 0°, 10°, 30°, 60°, 90° and 120° knee flexion angle and various values of the change in the joint component gap were calculated; 10°-0°, 30°-0°, 60°-0°, 90°-0° and 120°-0°. Multivariate regression analysis was used to determine the influencing of these parameters to post-operative knee flexion angle. RESULTS: The post-operative flexion angle was positively correlated with a joint component gap of 90° and 120° and the values of the change in the joint component gap of 90°-0° and 120°-0°. Multivariate regression analysis demonstrated pre-operative knee flexion angle, and the values of the change in the joint component gap of 90°-0° had a significant independent factor of post-operative knee flexion angle. CONCLUSIONS: Post-operative flexion angle is multi-factorial. However, it is important to avoid flexion component gap tightness as well as excessive flexion component gap looseness for acquisition of better flexion angle in CR TKA. LEVEL OF EVIDENCE: IV.