Rapid and simple isolation of precartilaginous stem cells: A novel approach without immunomagnetic bead sorting.

Objective: The present investigation was designed to devise a rapid and straightforward technique for the isolation of rat precartilaginous stem cells (PCSCs) that eschews the use of immunomagnetic bead sorting. Method: Rat neonates within 24 h of birth were selected for this study. Microsurgical techniques were used to harvest the femur, tibia, and the musculature of the knee joint. The ring of LaCroix between the metaphysis of the femur and the epiphysis was excised and divided into fragments of approximately 1 mm³. Tissue sections were cultured in Dulbecco's modified Eagle's medium(DMEM)/F12 medium supplemented with 20 % fetal bovine serum and 1 % penicillin-streptomycin. Cell digestion and passaging were performed using trypsin when cells reached 70%-80 % confluence. Third-generation cells underwent immunofluorescence staining and flow cytometry to evaluate fibroblast growth factor receptor-3(FGFR-3) and proliferating cell nuclear antigen(PCNA) expression, while ß-galactosidase staining was used to determine cellular senescence. Results: Within two days of isolation, numerous short spindle-shaped cells exhibiting distinct refractive properties were observed around the tissue fragments. These cells began to proliferate within 2-3 days and displayed ample cytoplasm. Adherent cells adopted various morphologies, including angular, triangular, and elongated spindles. By the fifth day, more than 80 % of the culture dish surface was covered with elongated cells, with some arranged in patterns reminiscent of whirlpools. Significant FGFR-3 and PCNA expression was confirmed via immunofluorescence in the third-generation cells. Additionally, flow cytometry identified that the proportion of cells positive for FGFR-3 and PCNA exceeded 98 %. Notably, the cells preserved their proliferative capacity through nine passages in vitro, with a marginal proportion showing senescence as indicated by ß-galactosidase staining alone. Conclusion: The developed tissue adherence protocol was used to successfully isolate PCSCs with positive FGFR-3 and PCNA expression, rendering the immunomagnetic bead sorting superfluous. The expression of FGFR-3 and PCNA in the isolated cells persisted through the ninth passage in vitro with minimal senescence.

Risk factors for unsuccessful reduction of chronic Monteggia fractures in children treated surgically.

Aims: To investigate the risk factors for unsuccessful radial head reduction (RHR) in children with chronic Monteggia fractures (CMFs) treated surgically. Methods: A total of 209 children (mean age 6.84 years (SD 2.87)), who underwent surgical treatment for CMFs between March 2015 and March 2023 at six institutions, were retrospectively reviewed. Assessed risk factors included age, sex, laterality, dislocation direction and distance, preoperative proximal radial metaphysis width, time from injury to surgery, reduction method, annular ligament reconstruction, radiocapitellar joint fixation, ulnar osteotomy, site of ulnar osteotomy, preoperative and postoperative ulnar angulation, ulnar fixation method, progressive ulnar distraction, and postoperative cast immobilization. Independent-samples t-test, chi-squared test, and logistic regression analysis were used to identify the risk factors associated with unsuccessful RHR. Results: Redislocation occurred during surgery in 48 patients (23%), and during follow-up in 44 (21.1%). The mean follow-up of patients with successful RHR was 13.25 months (6 to 78). According to the univariable analysis, time from injury to surgery (p = 0.002) and preoperative dislocation distance (p = 0.042) were identified as potential risk factors for unsuccessful RHR. However, only time from injury to surgery (p = 0.007) was confirmed as a risk factor by logistic regression analysis. Receiver operating characteristic curve analysis and chi-squared test confirmed that a time from injury to surgery greater than 1.75 months increased the rate of unsuccessful RHR above the cutoff (p = 0.002). Conclusion: Time from injury to surgery is the primary independent risk factor for unsuccessful RHR in surgically treated children with CMFs, particularly in those with a time from injury to surgery of more than 1.75 months. No other factors were found to influence the incidence of unsuccessful RHR. Surgical reduction of paediatric CMFs should be performed within the first two months of injury whenever possible.

Is there an alternative to the Delbet-Colonna classification? Introduction and reliability assessment of a new classification system for paediatric femoral neck fractures: preliminary results.

PURPOSE: The purpose of this study was to introduce a new classification system for paediatric femoral neck fractures (PFNFs) and to evaluate its reliability. METHODS: Two hundred and eight unilateral PFNFs (mean patient age: 9.0 ± 4.8 years) were included. Based on preoperative radiographs, the new classification system distinguished PFNFs without anterior or posterior translation (Type I), PFNFs with anterior (Type II) or posterior (Type III) translation, PFNFs with a comminuted medial or posterior column (Type IV), and subtrochanteric femoral fractures (SFFs; Type V). Radiographs were evaluated twice with an interval of two weeks by 19 raters with different specialties, experiences and geographical origins. The results were compared with a selection of 50 patient age-matched unilateral PFNFs and SFFs (mean patient age: 9.1 ± 4.9 years). These were graded twice by the same graders according to the Delbet-Colonna (D-C) classification. RESULTS: Four radiologists and 15 paediatric orthopaedic surgeons from Europe and Asia graded the radiographs. Fair agreement was found between radiologists (κ = 0.296 ± 0.01) and surgeons (κ = 0.3 ± 0.005) (P = 0.17), although more experienced surgeons performed better than less experienced ones; a similar fair assessment was found for raters from Europe (κ = 0.309 ± 0.021) and Asia (κ = 0.3 ± 0.006) and for type II, III and IV fractures; the κ value in the first evaluation (0.309) was similar to that in the second evaluation (0.298). The overall κ value of the D-C classification subtypes was significantly higher (0.599 ± 0.217) than that of the new classification, 0.326 ± 0.162 (t = 3.190 P = 0.005). CONCLUSIONS: The new classification system showed fair reliability relative to the D-C classification. The reliability of the new classification system was not affected by the specialty or geographic origin of the rater or the evaluation round, only by rater experience level. The concordance was worse for PFNFs with anterior or posterior translation or with a comminuted medial or posterior columns.

Diagnosis and treatment of "medial to lateral diagonal injury of the elbow" in children: Concomitant medial epicondylar and radial neck fractures.

Purpose: The simultaneous and ipsilateral occurrence of medial epicondylar and radial neck fractures is rare. This study evaluated the clinical and radiological outcomes of medial to lateral diagonal injury of the elbow (MELAINE). Methods: Six males and 6 females were diagnosed with MELAINE (left: 10, 83.3%; right: 2, 16.7%). Medial epicondylar and radial neck fractures were classified according to Papavasiliou's classification (seven type II, two type III, three type IV) and Judet's classification (three type I, four type II and five type III), respectively. All patients underwent surgery. The carrying angle, range of motion, and Kim et al. Elbow Performance Score were used to evaluate clinical and functional outcomes; related complications were recorded. Results: Mean age at injury and mean follow-up were 11.1 ± 2.5 (range, 6-14) and 40 ± 25.6 months (range, 13-90), respectively. All fractures consolidated in 6.3 ± 1.2 weeks on average (4-9). Outcomes were good (n = 1; 8.3%) to excellent (n = 11; 91.7%). The carrying angle of the injured and uninjured side was 15.5°± 2.6° and 14.7°± 2°, respectively (p = 0.218). The range of motion of elbow flexion-extension and forearm pronation-supination of the injured side was 144.2°± 10.4°, 4.6°± 5.4°, 76.7°± 9.1°, 80.4°± 9.2°, respectively, with no significant differences from the healthy side (p > 0.05). The Elbow Performance Score of the injured and uninjured side was 96.3 ± 5.3 and 98.8 ± 2.3, respectively (p = 0.139). No cases of infection, cubitus valgus, stiffness, or instability were recorded. Conclusion: Although uncommon, MELAINE should not be neglected. Surgery aims to stabilize the elbow and avoid valgus deformity. If diagnosed and treated, clinical and radiological results are excellent in most cases.

Causal Association Between Tea Consumption and Bone Health: A Mendelian Randomization Study.

Background: Much observational research reported that tea consumption decreases the risk of osteoarthritis (OA), rheumatoid arthritis (RA), and osteoporosis (OP) which are the three major bone disorders. However, the observed correlation is inconclusive. To determine the causal relationship between genetically predicted tea intake and OA, RA, and OP, we performed a two-sample Mendelian randomization (MR) study based on large samples. Methods: The European population's genome-wide association meta-analysis (GWAS) dataset identified SNPs associated with tea consumption was obtained from Neale Lab's analysis of UK Biobank data that comprised 349,376 participants of European ancestry. We extracted genetic data for knee OA (17,885 controls and 4,462 cases), hip OA (50,898 controls and 12,625 cases), and RA (43,923 controls and 14,361 cases) from the UK Biobank and OP cases (93083 controls and 1,175 cases) from FinnGen Data Freeze 2. A MR study was conducted to examine the effect of selected single nucleotide polymorphisms (SNPs) and OA, RA, and OP risk. Several sensitivity analyses were performed with weighted median and inverse-variance weighted methods for estimating the causal effects. Results: In this MR study, the genetically predicted per one cup increase of tea consumption was not associated with knee OA (OR 1.11,95% CI: 0.79-1.55) using IVW with random effect. Genetic predisposition to tea consumption was not associated with hip OA (OR: 1.20, 95% CI: 0.84-1.71), RA (OR: 1.24 95% CI: 0.81-1.91), and OP (OR: 1.11, 95% CI: 0.89, 1.39). Following the sensitivity analysis, there was no potential pleiotropy. Conclusion: According to our study, According to our study, there was no statistical power to confirm a causal relationship between tea consumption and the risk of knee OA, hip OA, RA, and OP.

Relationship Between Plasma Vitamin C and COVID-19 Susceptibility and Severity: A Two-Sample Mendelian Randomization Study.

Background: Considering the antioxidant function of Vitamin C, also called ascorbic acid, it is widely used against viral infections such as coronavirus disease (COVID-19) based on in vitro, observational, and ecological studies. Many confounding factors that can affect Vitamin C levels. Thus, the association described to date may not be causal. To determine the causal relationship between genetically predicted plasma Vitamin C and COVID-19 susceptibility and severity, we performed two-sample Mendelian randomization (MR) based on large samples. Methods: The summary-level data for Vitamin C was obtained from a GWAS meta-analysis, which included 52,018 individuals from four studies of European ancestry. Data for COVID-19 HGI results were obtained from the meta-analysis of 35 GWASs with more than 1,000,000 subjects of European ancestry, including 32,494 cases with COVID-19 susceptibility and 1,316,207 controls, 9,986 cases with COVID-19 hospitalization and 1,877,672 controls, and 5,101 cases with COVID-19 severe disease and 1,383,241 controls. Mendelian randomization (MR) analysis was conducted to examine the effect of selected single nucleotide polymorphisms and COVID-19 susceptibility, hospitalization, disease severity. Several sensitivity analyses were performed with inverse-variance weighted (random-effect model), inverse variance weighted (fixed-effect model), weighted median, and maximum likelihood methods for estimating the causal effects. Results: In this MR study, genetic predisposition to the levels of plasma Vitamin C was not associated with COVID-19 susceptibility (OR: 0.99, 95% CI: 0.84-1.17, P = 0.91), hospitalization (OR: 1.10, 95% CI: 0.71-1.71, P = 0.67) and severity (OR: 0.83, 95% CI: 0.43-1.59, P = 0.58). The association was consistent in complementary analyses. No potential heterogeneities and directional pleiotropies were observed for the analysis results. Conclusion: According to our study, no correlation was observed between plasma Vitamin C levels and COVID-19 susceptibility and severity. Further studies in different ethnics are necessary to explore the potential role and mechanisms of circulating serum Vitamin C levels on COVID-19.

Clinical, radiographic and pedobarographic analysis of skeletally immature patients with surgically treated distal metaphyseal fractures of the tibia: is concomitant fixation of the fibula necessary?

This study evaluated the outcomes of distal tibia metaphyseal fractures (DTMFs) managed surgically and compared the outcomes of patients with and without associated fibula fracture fixation. Thirty-two consecutive patients (14 males; mean age at the time of injury: 7.8 years) with closed displaced DTMFs, with (22 patients; group A) or without associated fibula fractures fixation (10 patients; group B), were included. Besides standard radiographic measurements, the following static and dynamic pedobarographic parameters were evaluated: foot total static plantar pressure percentage (PP%), static plantar pressure percentage of the forefoot (PP%) and of the rear foot (PP%), landing sequence of the metatarsals during contact with the ground (MT), and impulse percentage of the metatarsal heads (MT%) and the medial and lateral heel (MH% and LH%). All patients were followed for at least 2 years (range: 2-4.5 years). Functional outcomes were excellent to good in all but two patients (93.8%) at the last follow-up visit using the Johner-Wruths criteria'. Radiographic measurements, PP%, PP%, PP%, MT%, MH% and LH% were not significantly different between two groups as well as between injured and uninjured side of patients within the same group (P > 0.05). Abnormal MT sequence was found in 40.9% of group A (9/22) and in 40% of group B patients (4/10) (P > 0.05). Stabilization of associated fibula fractures did not significantly impact the clinical, radiographic and pedobarographic outcomes of the children with displaced DTMFs who were surgically treated. Level of evidence: Level III.

[Analysis of a multiple osteochondroma case caused by novel splice mutation (c.1164+1G to A) of EXT1 gene].

OBJECTIVE: To detect potential mutation of EXT1 gene in a pedigree affected with multiple osteochondroma and explore its pathogenic mechanism. METHODS: The coding regions and their flanking sequences of the EXT1/EXT2 genes were subjected to PCR amplification and Sanger sequencing. Suspected mutations were verified by excluding possible single nucleotide polymorphisms and bioinformatics analysis. Transcripts of the EXT1 gene in the proband were analyzed by TA clone-sequencing, with its abundance compared with that of healthy controls. RESULTS: DNA sequencing has identified in the proband a novel heterozygous point mutation (c.1164+1G to A) at the 5'splice sites of intron 3 of the EXT1 gene. The same mutation was not found in the healthy controls. Bioinformatics analysis indicated that the mutation is highly conserved and can lead to skipping of exon 3 or aberrant splicing. TA clone-sequencing indicated that the numbers of transcripts with skipping of exon 3 has significantly increased in the proband (< 0.05) compared with the controls. CONCLUSION: The c.1164+1G to A mutation has resulted in skipping of exon 3 in a proportion of EXT1 gene transcripts. As the result, the number of transcripts with tumor suppressing function is relatively reduced and has ultimately led to the tumors.