Unmodificated stepless regulation of CRISPR/Cas12a multi-performance.

As CRISPR technology is promoted to more fine-divided molecular biology applications, its inherent performance finds it increasingly difficult to cope with diverse needs in these different fields, and how to more accurately control the performance has become a key issue to develop CRISPR technology to a new stage. Herein, we propose a CRISPR/Cas12a regulation strategy based on the powerful programmability of nucleic acid nanotechnology. Unlike previous difficult and rigid regulation of core components Cas nuclease and crRNA, only a simple switch of different external RNA accessories is required to change the reaction kinetics or thermodynamics, thereby finely and almost steplessly regulating multi-performance of CRISPR/Cas12a including activity, speed, specificity, compatibility, programmability and sensitivity. In particular, the significantly improved specificity is expected to mark advance the accuracy of molecular detection and the safety of gene editing. In addition, this strategy was applied to regulate the delayed activation of Cas12a, overcoming the compatibility problem of the one-pot assay without any physical separation or external stimulation, and demonstrating great potential for fine-grained control of CRISPR. This simple but powerful CRISPR regulation strategy without any component modification has pioneering flexibility and versatility, and will unlock the potential for deeper applications of CRISPR technology in many finely divided fields.

Dynamic DNA Assembly by Programmable Hybridization Chain Reaction Mimicking Tubulin.

Dynamic biological structures involve the continual turnover of molecules within supramolecular assemblies such as tubulin. Inspired by dynamic biology self-organizing systems, we build an artificial dynamic structure based on DNA nanotechnology through a nonequilibrium chemical system. Herein, a metastable domain (MD), essentially a stem-loop structure, was introduced into DNA hairpins within hybridization chain reaction (HCR), thereby imparting dynamic activity to the DNA polymers. Hairpins with MD thermodynamically assemble to a high-energy polymer in the presence of trigger strands. The polymer can relax back to the stable unassembled state once the invader is added and finally relax to the activated hairpin by an anti-invader. Reversible assembly/disassembly of the HCR is achieved through invader/anti-invader cycles. We accomplished kinetic modulation, reversible conformational switching, cascading regulation, and enzyme activity control through the MD-HCR. We believe that the design of the MD-HCR could inspire the development of autonomous biological functions within artificial systems.

AND Logic-Gate-Based Dual-Locking Probe System for the Sensitive Detection of microRNA and APE1.

MicroRNA (miRNA) and apurinic/apyrimidinic endonuclease 1 (APE1) have been reported to be closely associated with cancers, making them potential crucial biomarkers and therapeutic targets. However, focusing on the detection of a single target is not conducive to the diagnosis and prognosis assessment of diseases. In this study, an AND logic-gate-based dual-locking hairpin-mediated catalytic hairpin assembly (DL-CHA) was developed for sensitive and specific detection of microRNA and APE1. By addition of a lock to each of the hairpins, with APE1 and microRNA serving as keys, fluorescence signals could only be detected in the presence of simultaneous stimulation by APE1 and miRNA-224. This indicated that the biosensor could operate as an AND logic gate. DL-CHA exhibited advantages such as a low background, rapid response, and high logic capability. Therefore, the biosensor serves as a novel approach to cancer diagnosis with significant potential applications.

Characterization of the CT-based risk factors for concomitant patellofemoral instability in patients with anterior cruciate ligament injury.

OBJECTIVES: Underestimation of concomitant patellofemoral instability in patients with anterior cruciate ligament (ACL) injury has aroused extensive attention. However, the characteristics of the combined injury is not well recognized. Hence, we aimed to characterize the features of the combined injury, and determine the radiographic risk factors. METHODS: Fifteen radiological parameters were identified after discussion and pilot-tested. Radiographic measurements were compared using the analysis of variance model with Tukey post hoc analysis. A stepwise binomial logistic regression was performed and a nomogram model combining the significant risk factors was created. The model performance was validated by C-index, calibration plot, and decision curve. RESULTS: A total of 204 patients (mean [SD] age, 25.1 [6.7] years; 108 [52.9%] male) were included. The final model was updated through regression analysis using 4 parameters as significant risk factors: lateral femoral condyle ratio (OR (95% CI), 1.194 (1.023 to 1.409)), medial anterior tibial subluxation (mATS) (OR (95% CI), 1.234 (1.065 to 1.446)), medial posterior plateau tibial angle (mPPTA) (OR (95% CI), 1.266 (1.088 to 1.500)), and trochlear depth (OR (95% CI), 0.569 (0.397 to 0.784)). C-index for the nomogram was 0.802 (95% CI, 0.731 to 0.873) and was confirmed to be 0.784 through bootstrapping validation. Calibration plot established a good agreement between prediction and observation. Decision curve analysis showed that if threshold probability was over 10%, using the nomogram adds more benefit than either all or none scheme. CONCLUSIONS: Lateral femoral condyle ratio, mATS, mPPTA, and trochlear depth are strong adverse predictors of patellofemoral instability in patients with ACL injury. CLINICAL RELEVANCE: This study characterizes the radiological features of the combined injury. Patellofemoral instability should be noted when treating ACL injuries. KEY POINTS: • The radiological characteristics of the combined ACL injury and patellofemoral instability is not well recognized. • Lateral femoral condyle ratio, mATS, mPPTA, and trochlear depth are predominant risk factors for patellofemoral instability in patients with ACL injury. • Patellofemoral instability should be noted when treating ACL injuries.

DNA-based molecular classifiers for the profiling of gene expression signatures.

Although gene expression signatures offer tremendous potential in diseases diagnostic and prognostic, but massive gene expression signatures caused challenges for experimental detection and computational analysis in clinical setting. Here, we introduce a universal DNA-based molecular classifier for profiling gene expression signatures and generating immediate diagnostic outcomes. The molecular classifier begins with feature transformation, a modular and programmable strategy was used to capture relative relationships of low-concentration RNAs and convert them to general coding inputs. Then, competitive inhibition of the DNA catalytic reaction enables strict weight assignment for different inputs according to their importance, followed by summation, annihilation and reporting to accurately implement the mathematical model of the classifier. We validated the entire workflow by utilizing miRNA expression levels for the diagnosis of hepatocellular carcinoma (HCC) in clinical samples with an accuracy 85.7%. The results demonstrate the molecular classifier provides a universal solution to explore the correlation between gene expression patterns and disease diagnostics, monitoring, and prognosis, and supports personalized healthcare in primary care.

A PAM-free CRISPR/Cas12a ultra-specific activation mode based on toehold-mediated strand displacement and branch migration.

CRISPR (clustered regularly interspaced short palindromic repeats) technology has achieved great breakthroughs in terms of convenience and sensitivity; it is becoming the most promising molecular tool. However, only two CRISPR activation modes (single and double stranded) are available, and they have specificity and universality bottlenecks that limit the application of CRISPR technology in high-precision molecular recognition. Herein, we proposed a novel CRISPR/Cas12a unrestricted activation mode to greatly improve its performance. The new mode totally eliminates the need for a protospacer adjacent motif and accurately activates Cas12a through toehold-mediated strand displacement and branch migration, which is highly universal and ultra-specific. With this mode, we discriminated all mismatch types and detected the EGFR T790M and L858R mutations in very low abundance. Taken together, our activation mode is deeply incorporated with DNA nanotechnology and extensively broadens the application boundaries of CRISPR technology in biomedical and molecular reaction networks.

Impact of sex and age on the lateralisation of the tibial tubercle in normal paediatric and adolescent populations.

PURPOSE: Numerous methods have been proposed to characterise tubercle lateralisation. However, their normal values and related changes remain unclear. Accordingly, it was aimed to determine the potential sex and age effects and determined the optimal individualised method of diagnosing lateralisation of the tibial tubercle in patients with recurrent patellar dislocation (RPD). METHODS: Measurements included the tibial tubercle-trochlear groove (TT-TG) distance, tibial tubercle-posterior cruciate ligament (TT-PCL) distance and tibial tubercle lateralisation (TTL); and the proximal tibial width (PTW), trochlear width (TW) and trochlear dysplasia index (TDI), for adjustment. A two-way analysis of variance was used to determine the effect of age, sex and their interaction within the normal group. When the age effect was statistically significant, a nonlinear regression was created. Areas under the receiver-operating characteristic curve (AUCs) were calculated to assess diagnostic accuracy. RESULTS: A total of 277 normal participants (mean [SD] age, 13.5 [2.6] years; 125 [45.1%] female) and 227 patients with RPD (mean [SD] age, 13.5 [2.6] years; 161 [58.1%] female) were analysed. It was found that in the normal group, in patients aged 7-10, TT-PCL distance (p = 0.006), TTL (p = 0.007) and TT-PCL/PTW (p < 0.001) were significantly larger in females than in males. A significant sex effect was also detected on TT-TG/TW (p = 0.014). TT-TG distance, TT-PCL distance, TTL and TT-PCL/PTW (in male patients) approached an established normal adult value of 12.3 mm, 20.9 mm, 0.64 and 0.28, respectively, with increasing age (p < 0.001). The AUC was greater for TT-TG/TDI and TT-TG/TW (p ≤ 0.01) and TT-TG/TDI outperformed TT-TG/TW in patients aged 15-18 (p = 0.004). CONCLUSIONS: Tubercle lateralisation increased with age and was affected by sex, with the exception of TT-TG distance and TT-TG/TDI. TT-TG/TDI is the optimal method of diagnosing a lateralized tibial tubercle in patients with RPD. These findings assist with the evaluation of tubercle lateralisation in that they provide a proper protocol for paediatric and adolescent populations with RPD; and thus, will help determine whether medial tubercle transfer should be included among the tailored surgical procedures considered for the treatment of patients with RPD. LEVEL OF EVIDENCE: Level III.

RART-LAMP: One-Step Extraction-Free Method for Genotyping within 40 min.

Loop-mediated isothermal amplification (LAMP) is a commonly used alternative to PCR for point-of-care detection of nucleic acids due to its rapidity, sensitivity, specificity, and simpler instrumentation. While dual-labeled TaqMan probes are widely used in PCR for single-nucleotide polymorphism (SNP) genotyping, real-time LAMP primarily relies on turbidimetry or intercalator fluorescence measurements, which can be non-specific and generate false-positive results. In this study, we propose a closed-tube, dual-labeled RNA-modified probes and RNase H II-assisted real-time LAMP (RART-LAMP) method for SNP genotyping. Our findings indicate that (1) fluorescence signals were predominantly derived from probe hydrolysis rather than hybridization, (2) temperature-controlled hybridization between the probe and template ensured the specificity of SNP analysis, and (3) RNase H II hydrolysis between the target containing SNP sites and probes did not exhibit sequence specificity. Our RART-LAMP approach demonstrated excellent performance in genotyping C677T clinical samples, including gDNA extracted from blood, saliva, and swabs. More importantly, saliva and swab samples could be directly analyzed without any pretreatment, indicating promising prospects for nucleic acid analysis at the point of care in resource-limited settings.

A Spatially Controlled Proximity Split Tweezer Switch for Enhanced DNA Circuit Construction and Multifunctional Transduction.

DNA strand displacement reactions are vital for constructing intricate nucleic acid circuits, owing to their programmability and predictability. However, the scarcity of effective methods for eliminating circuit leakages has hampered the construction of circuits with increased complexity. Herein, a versatile strategy is developed that relies on a spatially controlled proximity split tweezer (PST) switch to transduce the biomolecular signals into the independent oligonucleotides. Leveraging the double-stranded rigidity of the tweezer works synergistically with the hindering effect of the hairpin lock, effectively minimizing circuit leakage compared with sequence-level methods. In addition, the freely designed output strand is independent of the target binding sequence, allowing the PST switch conformation to be modulated by nucleic acids, small molecules, and proteins, exhibiting remarkable adaptability to a wide range of targets. Using this platform, established logical operations between different types of targets for multifunctional transduction are successfully established. Most importantly, the platform can be directly coupled with DNA catalytic circuits to further enhance transduction performance. The uniqueness of this platform lies in its design straightforwardness, flexibility, scalable intricacy, and system compatibility. These attributes pave a broad path toward nucleic acid-based development of sophisticated transduction networks, making them widely applied in basic science research and biomedical applications.

Patella-Posterior Turning Point of the Distal Femur Distance Is a Potential Indicator for Diagnosing Patella Alta in Recurrent Patellar Dislocation Population.

PURPOSE: To introduce a simple patellar height measurement method (patella-posterior turning point of the distal femur [P-PTP] Distance) independent of patellar anatomy with standardized patient position, and tested the reliability, validity, and diagnostic accuracy compared with commonly used ratios in knee extension. METHODS: We retrospectively reviewed 418 computed tomography (CT) images of the knee joint in a group of patients who were diagnosed recurrent patellar dislocation (RPD). With the three-dimensional (3D) CT reconstructed knee, patellar height was qualitatively assessed by the patellar engagement with the femoral trochlea in terminal knee extension to divide RPD population into case (patella alta) and control group. With digitally reconstructed lateral radiographs, patellar height was measured with P-PTP distance (perpendicular distances between the distal edge of patella articular surface and posterior turning point of distal femur), and four commonly used ratios: Caton-Deschamps index, Modified Insall-Salvati index, Blackburne-Peel index, and Insall-Salvati index. An unpaired t-test was conducted to determine significant differences between groups. Correlation coefficient, intra- and inter-observer reliability, receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) were also calculated. RESULTS: 198 knee images (198 patients) were included for final evaluation. Patella alta was present in 72 knees (36.3%) with RPD. The effect size was relatively large between the case and control group for P-PTP distance (d = -1.619; 95% CI, -1.948 to -1.286). P-PTP distance correlated moderately to strongly with four commonly used ratios (P < .001). Intraobserver and interobserver reliability was good for P-PTP distance. The AUC of the ROC curve was categorized as excellent for P-PTP distance, better than other measurements (P < .001), and the cutoff value was 4.2 mm with the highest sensitivity (86.11%) and specificity (84.92%). CONCLUSIONS: The measurement method, P-PTP distance, showed good intra-observer and inter-observer reliability, well correlated with commonly used ratios, and presented best diagnostic accuracy among commonly used ratios for predicting RPD. P-PTP distance might be a potential indicator for identifying patella alta in RPD patients when supine and knee extended. CLINICAL RELEVANCE: The measurement reported in this study may help in advancing clinical evaluation of patella alta, providing an alternative and simple method to measure patellar height. Standing or weight-bearing plain lateral radiographs obtained from the routine practice should be further assessed in the next step to further validate the method.

Sulcus depth, congruence angle, Wiberg index, TT-TG distance, and CDI are strong predictors of recurrent patellar dislocation.

PURPOSE: To characterize patellofemoral morphology, determine radiological cutoff values based on computed tomography (CT) images, and clarify predominant anatomic risk factors in young patients with recurrent patellar dislocation (RPD). METHODS: A total of 212 knees (age 23.0 ± 5.3 years, female: male = 110:102) with CT-confirmed RPD were enrolled for analysis. The control group was matched by age and sex (2:1). Patellofemoral and trochlear morphology, patellofemoral alignment, and other parameters were assessed by CT. Standardized methods were used for measurements. Receiver operating characteristic (ROC) curves and the areas under the ROC curve (AUCs) were used to assess accuracy in diagnosing RPD. The index of union (IU) was employed to identify a cutoff value for risk. A binary logistic regression model (Forward, LR) was constructed, and standardized coefficients were calculated to compare the relative strength of different predictors. Factors included in the model were employed to calculate distributions in the studied population. RESULTS: The majority of measurements showed a significant difference between the RPD and control groups when comparing mean values. The AUC value was favorable for sulcus depth and significantly better than that for other trochlear morphology parameters (P < 0.0001). Cutoff values were 0.58 for the Wiberg index, 153.3° for the sulcus angle, 3.6 mm for the sulcus depth, 1.94 for the trochlear width ratio, 16.2° for the lateral trochlear inclination, 17.2° for the patellar lateral tilt, 25.5° for the congruence angle, 1.2 for the patellar height [Caton-Deschamps index (CDI)], 6.1° for the knee rotation, and 16.4 mm for the tibial tubercle-trochlear groove (TT-TG) distance. A binary logistic regression model (χ2 = 20.826, P = 0.008) including the Wiberg index, sulcus depth, congruence angle, patellar height (CDI), and TT-TG distance was constructed for RPD (P ≤ 0.015), which showed excellent diagnostic performance, as indicated by an AUC of 0.989 (95% CI, 0.979 to 0.998). Sulcus depth showed the strongest relationship with RPD, followed by the congruence angle and Wiberg index, as indicated by the standardized coefficients - 1.43, 1.02, and 0.97, respectively. Eighty-seven percent of the RPD population had sulcus depth ≤ 3.6 mm. CONCLUSIONS: This study provides a systematic reference of cutoff values based on CT data. The combination of five anatomic risk factors (sulcus depth, congruence angle, Wiberg index, TT-TG distance, and CDI) may be an excellent predictor of RPD as initial risk assessment. Sulcus depth, as the most frequently observed anatomic risk factor, showed the best diagnostic performance among trochlear morphological parameters and the strongest relationship with RPD, followed by the congruence angle and Wiberg index. In clinical practice, the sulcus depth, congruence angle, Wiberg index, TT-TG distance, and CDI with the proposed cutoff values can be used in combination to evaluate RPD. When assessing trochlear morphology, sulcus depth is recommended as the best radiological parameter. LEVEL OF EVIDENCE: Level III.

Patellar shift ratio (PSR) is the optimal measurement for characterising lateral patellar shift and a reliable predictor of recurrent patellar dislocation.

PURPOSE: To propose a new measurement, the patellar shift ratio (PSR), for characterising lateral patellar shift; to determine its diagnostic accuracy in predicting recurrent patellar dislocation (RPD); and to identify the effect of patellar tilt and morphology on shift measurements. METHODS: Variables including the PSR, bisect offset (BSO), BSO adjusted by trigonometric analysis, linear patellar displacement (LPD) distance and congruence angle were measured on axial computed tomography (CT). Furthermore, PSR was classified into grades determined by the threshold generated by receiver operating characteristic (ROC) curves. Youden's index was used to identify the ideal threshold on the curve. The area under the ROC curve (AUC) values and likelihood ratios were calculated to assess diagnostic performance in predicting RPD. Correlation analysis was conducted to identify the effects of lateral tilt and the Wiberg index on lateral shift. Intra- and interobserver reliability were evaluated using the intraclass correlation coefficient. RESULTS: The study included 54 RPD patients and 54 controls matched by sex and age. The threshold for PSR was 24.3% (sensitivity, 96.30%; specificity, 85.19%). AUCs were categorised as excellent for all protocols (P < 0.001). The positive likelihood ratio of the PSR for predicting RPD was 6.50. PSR was categorised in terms of lateral trochlear quadrants consisting of 25% increments. Lateral patellar tilt showed a strong positive correlation with BSO (P < 0.001); moderate correlations with PSR, LPD distance and adjusted BSO (P ≤ 0.001); and a weak correlation with congruence angle (P = 0.034). The Wiberg index presented a positive moderate correlation with BSO (P < 0.001), while no correlation was found with PSR, LPD distance, or congruence angle (n.s.). All parameters showed good repeatability (intraobserver) and reproducibility (interobserver). CONCLUSIONS: The PSR showed excellent diagnostic performance as indicated by likelihood ratios, measurement accuracy (being relatively resistant to the effects of tilt and the Wiberg index), and intra- and interobserver reliability. The optimal PSR threshold for predicting RPD risk was 24.3%, and the grade of PSR (normal, 1-4) was also a reliable and easily calculated predictor of RPD. This quantifying method to characterise lateral patellar shift is an alternative useful method for clinical assessments of RPD patients and for research on patellofemoral congruence. LEVEL OF EVIDENCE: Level III.

Rotator cuff tear reaching the superior half portion of the humeral head causes shoulder abduction malfunction.

PURPOSE: To examine the biomechanical properties governing posterosuperior rotator cuff (RC) tear progression and dynamic shoulder abduction function, in the absence of excess loading. METHODS: Twelve freshly frozen cadaveric shoulders were evaluated via an established dynamic shoulder abduction stimulator. The shoulder abduction functions were primarily evaluated using subacromial contact pressure (SACP) during an abduction procedure, and subsequent middle deltoid force (MDF) under 5 conditions: (1) intact, (2) anterior 1/3 posterosuperior rotator cuff (PSRC) tear, (3) anterior 2/3 PSRC tear, (4) entire PSRC tear, and (5) global RC tear (tear involving the entire superior RC). RESULTS: No obvious differences were observed in the peak MDF required for abduction, and in the peak SACP among the four PSRC tear statuses (49.8 ± 9.2 N, 0.39 ± 0.05 mPa [1/3 PSRC tear]; 49.3 ± 6.8 N, 0.40 ± 0.06 mPa [2/3 PSRC tear]; 51.6 ± 7.0 N, 0.44 ± 0.08 mPa [entire PSRC tear]), as well as intact statuses (48.3 ± 9.8 N, 0.40 ± 0.05 mPa). However, significant elevations in the peak MDF and peak SACP levels were observed among the four PSRC tear statuses and global RC tear (68.1 ± 9.3 N; 4.12 ± 1.50 mPa, P < 0.01). CONCLUSION: In the absence of excess loading, the biomechanical function of the shoulder was not impaired by a simple PSRC tear. However, once the tear size reached the half superior portion of the humeral head, the humeral head migrated to the surface of the subacromion, and this action markedly decreased shoulder abduction function.

Meniscal resection increases the risk of residual knee laxity even in patients undergoing anatomic double-bundle anterior cruciate ligament reconstruction with eight strands of hamstring autografts.

PURPOSE: To compare the midterm clinical outcomes of different meniscal surgeries in patients undergoing anatomic double-bundle anterior cruciate ligament reconstruction (DB-ACLR) with eight strands of hamstring (HT8) autografts and explore the potential predictive risk factors for residual knee laxity. METHODS: From 2010 to 2017, a total of 410 patients who underwent anatomic trans-tibial DB-ACLR with HT8 autografts (169 patients without meniscal surgery, 105 patients with meniscal repair, and 136 patients with meniscal resection) were included in this study. The equivalent graft diameter was introduced to make the total graft size of DB-ACLR comparable with that of single-bundle ACLR and calculated as the square root of the quadratic sum of the diameter for each bundle. Residual laxity was defined as excessive anterior tibial translation or residual pivot shift at any follow-up visit, while graft rupture was confirmed by second-look arthroscopy or magnetic resonance imaging. RESULTS: The mean follow-up period was 8.3 ± 2.2 years. The mean equivalent graft diameter was 9.9 ± 0.7 mm. Graft rupture was confirmed in 16 (3.9%) patients, while residual laxity was detected in 72 (17.6%) patients (34 [25.0%] in the meniscal resection group vs. 22 [13.0%] in the no meniscal surgery group, p = 0.021). In the multivariate logistic regression analysis, high-grade preoperative knee laxity (odds ratio OR 2.04, p = 0.020), equivalent graft diameter < 9 mm (OR 3.31 compared with 9-10 mm, p = 0.012; OR 3.28 compared with ≥ 10 mm, p = 0.019), and meniscal resection (OR 1.94 compared with no meniscal surgery, p = 0.045) were associated with residual laxity. CONCLUSION: During a midterm follow-up, meniscal resection increased the risk of residual knee laxity even in patients undergoing anatomic DB-ACLR with HT8 autografts. Increasing the hamstring graft diameter and preserving the menisci are important strategies for ACLR to restore knee stability. LEVEL OF EVIDENCE: Level III.

Dynamic Double-Sling Augmentation Prevents Anteroinferior Translation for Recurrent Anteroinferior Shoulder Dislocation With 20% Glenoid Bone Loss: A Cadaveric Biomechanical Study.

PURPOSE: To biomechanically compare the dynamic double-sling with single-sling augmentation using the conjoined tendon (CT) with 20% of an anteroinferior glenoid bone defect under the high loads in shoulders. METHODS: With the shoulder in 60° of glenohumeral abduction and 60° of external rotation, the 12 shoulders stability was tested sequentially in 5 conditions: intact, 20% glenoid bone loss, Bankart repair, single-sling augmentation with the CT, and double-sling augmentation with both the CT and long head of the biceps tendon (LHBT). The anteroinferior humeral head (HH) translation force of 20N, 30N, 40N, 50N, or 60N was applied to determine the shoulder stability in each condition. RESULTS: The total HH translation over 8.77 mm represented the anteroinferior shoulder instability (95% confidence interval of bone defect: 7.76-8.77 mm). A significant increase in anteroinferior HH translation was demonstrated after the creation of 20% glenoid bone defect under the 20N translational force (10.52 ± 0.71 mm). Structural failure after the Bankart repair and the single-sling augmentation under the 30N (9.84 ± 1.25 mm) and 40N (9.59 ± 0.66 mm) translational forces, respectively, were observed. The double-sling augmentation effectively prevented the anteroinferior HH translation under the translational force of less than 40N, and only half of the augmentation structure (8.25 ± 1.66 mm) had failed under the 50N translational forces. CONCLUSION: In the absence of any Hill-Sachs lesion and when tested at 60° abduction and external rotation in shoulders with 20% glenoid bone defects, at time-zero, the double-sling augmentation strategy could effectively prevent anteroinferior translation when compared with the Bankart repair or the single-sling augmentation technique under all magnitudes of the translational force in biomechanical simulation. Nevertheless, none of the constructs restored the HH translation to the normal intact state. CLINICAL RELEVANCE: Double-sling augmentation technique may represent a reliable option for preventing anteroinferior translation.

Differentiation Ability of Tendon-Derived Stem Cells and Histological Characteristics of Rotator Cuff Remnant on the Greater Tuberosity Degenerated With Age and Chronicity.

PURPOSE: To explore and measure the presence and activity of tendon-derived stem cells (TDSCs), as well as histological changes of rotator cuff remnant by age and chronicity of the rotator cuff tear (RCT). METHODS: 154 patients with a full-thickness tear of supraspinatus and/or infraspinatus tendon were included. 52 qualified remnants of the greater tuberosity were captured through arthroscopy. TDSCs in the remnants were isolated for proliferation ability, basal gene expression, and trilineage differentiation detection. Histological characteristics were evaluated by observation of staining under a light microscope and transmission electron microscopy (TEM). To observe the effect of age, samples were divided into two groups: young (＜60 years old) and old (≥60 years old). For chronicity comparison, samples were divided into three groups: acute group (＜3 months), intermediate group (3-12 months), and chronic group (≥12 months). RESULTS: Between age groups, the remnants in older patients were found to have lower TDSC proliferation ability (cell counting kit-8 results, old: .5325 ± .050, young: .6623 ± .196; P = .008) and basal expression of aggrecan (.630 ± .239; P = .002) and TGF-ß1 (transforming growth factor-ß1, .589 ± .326, P = .008), weaker ability of chondrogenic differentiation. Furthermore, the remnant tendons in chronic group was found to have weaker adipogenic and chondrogenic differentiation ability of TDSCs, lower tendon degenerative score (acute: 3.57 ± 1.902, intermediate: 5.94 ± 2.313, chronic: 6.86 ± 2.193; P = .023), increased type III collagen region ratio in insertion area (acute: 86.10% ± 8.29%, intermediate: 94.06% ± 5.36%, chronic: 98.90% ± .49%; P = .023), and larger fibril diameters. CONCLUSION: Differentiation ability of TDSCs derived from the rotator cuff remnant was reduced with age and chronicity. Histological degeneration of remnant tendon deteriorated with chronicity. Remnant in the greater tuberosity was still alive, but those in young or acute injury patients were more active after full-thickness RCT. CLINICAL RELEVANCE: TDSCs exist in rotator cuff remnant on the greater tuberosity and have multilineage differentiation ability. But the remnant degenerated with age and chronicity.

Bankart Repair With Transferred Long Head of the Biceps Provides Better Biomechanical Effect Than Conjoined Tendon Transfer in Anterior Shoulder Instability With 20% Glenoid Defect.

PURPOSE: To examine the biomechanical differences between labral repair with transferred conjoined tendon and transferred long head of the biceps tendon (LHBT) for anterior shoulder instability with 20% bone loss. METHODS: Twelve cadaveric shoulders were tested in sequent 5 conditions: intact, 20% glenoid defect, Bankart repair, Bankart repair with transferred conjoined tendon (dynamic conjoined tendon sling, DCS), and with transferred LHBT (dynamic LHBT sling, DLS) at 60° of glenohumeral abduction and 60° of external rotation. The physiological glenohumeral joint load was created by forces applied to the rotator cuff, conjoined tendon, and LHBT. The glenohumeral compression force and range of motion were recorded before anteroinferior force application. The anterior, inferior, and total translations were measured with 20, 30, 40, and 50 N of anteroinferior force, respectively. RESULTS: Anteroinferior glenoid defect led to significant increase of humerus translation and decrease of glenohumeral compression force. DLS provided better resistance effect in both anterior-posterior and superior-inferior directions than DCS under high loading condition (40 N, P =.03; 50 N, P <.01). Both DCS and DLS procedures could further restore glenohumeral compression force with Bankart repair (Bankart repair: 32.1 ± 4.0 N; DCS: 36.7 ± 3.2 N, P < .01; DLS: 35.8 ± 3.6 N, P =.03). No range of motion restrictions were observed relative to the normal shoulder. CONCLUSIONS: Both the DLS and DCS techniques could reduce the anterior-inferior translation and partially restore the glenohumeral stability in anterior shoulder instability with 20% anteroinferior glenoid defect compared with Bankart repair. Under greater loading conditions, DLS provides better stability than DCS. CLINICAL RELEVANCE: Shoulder stability can be restored by DLS and DCS with low load. With greater shoulder stability requirements, DLS might be a better option than DCS for anterior shoulder instability with 20% bone loss.

Steep lateral tibial slope measured on magnetic resonance imaging is the best radiological predictor of anterior cruciate ligament reconstruction failure.

PURPOSE: To identify the radiological predictive risk factors for anterior cruciate ligament reconstruction (ACLR) failure, compare the diagnostic accuracies of different parameters of conventional radiographs and magnetic resonance imaging (MRI), and determine the cutoff values for patients at higher risk. METHODS: Twenty-eight patients who were diagnosed as ACLR failure via MRI or arthroscopic examination were included in the study group. They were matched to 56 patients who underwent primary ACLR with the same surgical technique and without graft failure at the minimum 24-month follow-up by age, sex, and body mass index. On true lateral whole-leg radiographs, the posterior tibial slope (PTS) referenced to the tibial mechanical axis (PTS-mechanical), PTS referenced to the tibial proximal anatomical axis (PTS-anatomical), and anterior tibial translation (ATT) were measured. On the sagittal slices of MRI, the medial tibial slope (MTS), medial tibial plateau (MTP) subluxation (MTPsublx), lateral tibial slope (LTS), and lateral tibial plateau (LTP) subluxation (LTPsublx) were obtained. Receiver operator characteristic (ROC) curves were constructed to compare the diagnostic performance and determine the cutoff values of different radiological parameters. RESULTS: The study group demonstrated higher values of PTS-mechanical (10.7° ± 2.9° vs 8.7° ± 1.9°, p = 0.003), PTS-anatomical (13.2° ± 2.8° vs 10.5° ± 2.5°, p < 0.001), ATT (10.7 ± 3.3 mm vs 8.9 ± 2.2 mm, p = 0.014), LTS (9.4° ± 2.1° vs 5.5° ± 2.5°, p < 0.001), and LTPsublx (8.2 ± 2.8 mm vs 6.8 ± 1.9 mm, p = 0.009) as compared with the control group. The area under the ROC curve of LTS was significantly larger than that of PTS-mechanical (p = 0.006) and PTS-anatomical (p = 0.020). Based on the maximum Youden indexes, the cutoff values of PTS-mechanical, PTS-anatomical, and LTS were 10.1° (sensitivity, 64.3%; specificity, 78.6%), 12.0° (sensitivity, 71.4%; specificity, 71.4%), and 7.7° (sensitivity, 85.7%; specificity, 80.4%), respectively. CONCLUSION: Due to the morphological asymmetry of the MTP and LTP, steep LTS measured on MRI is the best radiological predictor of ACLR failure. Detailed measurement of the LTS on MRI is recommended to evaluate the risk of ACLR failure prior to the surgery. LEVEL OF EVIDENCE: III.

Allograft contamination during suture preparation for anterior cruciate ligament reconstruction: an ex vivo study.

PURPOSE: Effects of suture preparation on graft contamination remain unknown in anterior cruciate ligament reconstruction (ACLR). This study aimed to evaluate the incidence of allograft contamination at different time points of graft preparation and investigate differences in contamination between different sites of the allografts. METHODS: Fourteen hamstring tendon (HT), 9 quadriceps tendon (QT), and 9 bone-patellar tendon-bone (BTB) allografts were harvested, sterilised, and stored following routine procedures. Graft suture preparation was performed with baseball stitching for soft tissue and bone drilling for bone plug. The time was recorded simultaneously. The graft was kept moist in a standard operating room environment for 30 min after the initiation of preparation. The specimens were obtained from the middle and both ends of each graft for culture at three different time points: pre-suturing, post-suturing, and 30 min after the initiation of preparation. A total of 192 specimens were transferred to the microbiology laboratory for culture, identification, and semi-quantitative assessment. Culture results were classified as negative, poor, and abundant based on the extent of growth. Contamination level was recorded as low or high corresponding to culture results of poor or abundant. RESULTS: The duration of suture preparation was 348, 301, and 246 s for HT, QT, and BTB (P = 0.090). The specimens had a positive culture rate of 41/192 (21.4%), of which 21 were from the ends and 20 from the middle. More positive samples with abundant bacterial growth were detected from the ends than from the middles post-suturing (7/8 vs. 1/7, P = 0.010) and at 30 min (6/11 vs. 0/11, P = 0.012). The total graft contamination rate was significantly higher at 30 min (19/32, 59.4%) than pre-suturing (4/32, 15.6%) and post-suturing (9/32, 28.1%) (P < 0.001). The contamination rate with abundant bacterial growth was higher post-suturing (7/32, 21.9%) than pre-suturing (0%). No statistically significant differences were found among the three types of allografts. CONCLUSION: The contamination rate increases significantly at 30 min compared with pre-suturing and post-suturing. Suture preparation may have introduced the high-level contamination, to which the ends of the graft were more prone than the middle. Therefore, routine prophylactic decontamination after suture preparation should be considered, especially for the ends of the grafts.

Xeno nucleic acid probes mediated methylation-specific PCR for single-base resolution analysis of N6-methyladenosine in RNAs.

Reliable and cost-effective quantification of RNA modifications at a specific gene locus is essential to elucidate the pathogenic mechanism encoded by RNA epigenetics. Current methods to quantify N6-methyladenosine (m6A) at specific sites can hardly satisfy the requirement of clinical application because epigenetic information is easily lost through polymerase chain reaction (PCR) assay or other isothermal amplification methods unless tedious pretreatment is applied. Herein, we propose a simple xeno nucleic acid (XNA) as a blocker probe to mediate the methylation specific reverse transcription quantitative polymerase chain reaction (MsRT-qPCR) assay to directly magnify the minor differences between epigenetic bases and unmodified bases in RNA. Strand displacement reactions selectively initiated between the reverse transcription primer (RT-primer) and the XNA probe at the m6A template given the affinity differences between the blocker probes and the m6A-modified RNA (m6A-RNA) and unmodified RNA (A-RNA). Thus, preferential amplification of m6A-RNA was allowed. Integration of a well-established oligo-modified Fe3O4@UiO-66-NH4 allowed purification of mRNA and lncRNA from cellular total RNA samples and greatly reduced the non-specific interference of m6A detection in real samples. Multiple specific sites of m6A in mRNA and lncRNA samples are also successfully quantified. The XNA probe-based m6A assay required only common and available lab equipment and materials, which can be applied in m6A-related fundamental studies and clinical diagnosis.