Epigenetic control of gene expression is critical for normal fetal development. However, chromatin-related mechanisms that activate bone-specific programs during osteogenesis have remained underexplored. Therefore, we investigated the expression profiles of a large cohort of epigenetic regulators (>300) during osteogenic differentiation of human mesenchymal cells derived from the stromal vascular fraction of adipose tissue (AMSCs). Molecular analyses establish that the polycomb group protein EZH2 (enhancer of zeste homolog 2) is down-regulated during osteoblastic differentiation of AMSCs. Chemical inhibitor and siRNA knockdown studies show that EZH2, a histone methyltransferase that catalyzes trimethylation of histone 3 lysine 27 (H3K27me3), suppresses osteogenic differentiation. Blocking EZH2 activity promotes osteoblast differentiation and suppresses adipogenic differentiation of AMSCs. High throughput RNA sequence (mRNASeq) analysis reveals that EZH2 inhibition stimulates cell cycle inhibitory proteins and enhances the production of extracellular matrix proteins. Conditional genetic loss of Ezh2 in uncommitted mesenchymal cells (Prrx1-Cre) results in multiple defects in skeletal patterning and bone formation, including shortened forelimbs, craniosynostosis, and clinodactyly. Histological analysis and mRNASeq profiling suggest that these effects are attributable to growth plate abnormalities and premature cranial suture closure because of precocious maturation of osteoblasts. We conclude that the epigenetic activity of EZH2 is required for skeletal patterning and development, but EZH2 expression declines during terminal osteoblast differentiation and matrix production.

Background: Clinical translation of mesenchymal stromal cells (MSCs) necessitates basic characterization of the cell product since variability in biological source and processing of MSCs may impact therapeutic outcomes. Although expression of classical cell surface markers (e.g., CD90, CD73, CD105, and CD44) is used to define MSCs, identification of functionally relevant cell surface markers would provide more robust release criteria and options for quality control. In addition, cell surface expression may distinguish between MSCs from different sources, including bone marrow-derived MSCs and clinical-grade adipose-derived MSCs (AMSCs) grown in human platelet lysate (hPL). Methods: In this work we utilized quantitative PCR, flow cytometry, and RNA-sequencing to characterize AMSCs grown in hPL and validated non-classical markers in 15 clinical-grade donors. Results: We characterized the surface marker transcriptome of AMSCs, validated the expression of classical markers, and identified nine non-classical markers (i.e., CD36, CD163, CD271, CD200, CD273, CD274, CD146, CD248, and CD140B) that may potentially discriminate AMSCs from other cell types. More importantly, these markers exhibit variability in cell surface expression among different cell isolates from a diverse cohort of donors, including freshly prepared, previously frozen, or proliferative state AMSCs and may be informative when manufacturing cells. Conclusions: Our study establishes that clinical-grade AMSCs expanded in hPL represent a homogeneous cell culture population according to classical markers,. Additionally, we validated new biomarkers for further AMSC characterization that may provide novel information guiding the development of new release criteria. Clinical trials: Use of Autologous Bone Marrow Aspirate Concentrate in Painful Knee Osteoarthritis (BMAC): Clinicaltrials.gov NCT01931007. Registered August 26, 2013. MSC for Occlusive Disease of the Kidney: Clinicaltrials.gov NCT01840540. Registered April 23, 2013. Mesenchymal Stem Cell Therapy in Multiple System Atrophy: Clinicaltrials.gov NCT02315027. Registered October 31, 2014. Efficacy and Safety of Adult Human Mesenchymal Stem Cells to Treat Steroid Refractory Acute Graft Versus Host Disease. Clinicaltrials.gov NCT00366145. Registered August 17, 2006. A Dose-escalation Safety Trial for Intrathecal Autologous Mesenchymal Stem Cell Therapy in Amyotrophic Lateral Sclerosis. Clinicaltrials.gov NCT01609283. Registered May 18, 2012.

Rthroplasty technique for the treatment of inflammatory and posttraumatic arthritis of the elbow....

Study Design. A retrospective cohort study reporting the use of intraoperative computed tomography (CT) and image-guided navigation system for the placement of pedicle screws in pediatric compared with adult patients. Objective. To evaluate the accuracy of open pedicle screw placement in pediatric patients using intraoperative CT and 3-dimensional (3D) image-guided navigation. Summary of Background Data. Pedicle screws are widely used in children for the correction of spinal deformity. Navigation systems and intraoperative CT are now available as an adjunct to fluoroscopy and anatomic techniques for placing pedicle screws and verifying screw position. Methods. From 2007 to 2010, 984 pedicle screws were placed in a consecutive series cohort of 50 pediatric patients for spinal deformity correction with the use of intraoperative CT (O-arm, Medtronic, Inc, Louisville, CO) and a computerized navigation system (Stealth, Medtronic, Inc, Louisville, CO). The primary outcome measure for this study is redirection or removal of screw on the basis of the intraoperative CT imaging. During the study period, 1511 screws were placed in adult patients using the same image guidance system. Results. A total of 984 pedicle screws were implanted using real-time navigation, with a mean of 20 screws per patient (range: 2–34). On the basis of intraoperative CT, 35 screws (3.6%) were revised (27 redirected and 8 removed), representing a 96.4% accuracy rate. No patients returned to the operating room because of screw malposition. Of the 1511 screws placed in adult patients, 28 (1.8%) were revised intraoperatively for malposition on CT imaging, for an overall 98.2% accuracy rate. Screw revision thus was more common in the pediatric population (P = 0.008). However, the pediatric screw accuracy rate is significantly higher than the findings from a recent meta-analysis of predominantly nonnavigated screws in children, reporting a 94.9% accuracy rate (P = 0.03). Conclusion. We report 96.4% accuracy in pediatric pedicle screw placement using intraoperative CT and a 3D navigation system. This is similar to other reports and has better accuracy than a recent meta-analysis of nonnavigated screws in children.

Background: Long-term studies have indicated good outcomes for most patients with Legg-Calvé-Perthes disease. However, clinical experience suggests that less favorable outcomes are common. We sought to prospectively document pain and function in a cohort of adults who had previously been treated nonoperatively for Legg-Calvé-Perthes disease. Methods: Patients in our region with Legg-Calvé-Perthes disease were enrolled between 1984 and 1991 as part of a multicenter prospective trial and were treated with hip range-of-motion exercises or bracing. Patients returned for physical examination, radiographs, and completion of outcome measures including the Nonarthritic Hip Score (NAHS) and the Iowa Hip Score (IHS). Results: Fifty-six patients (fifty-eight hips) were examined at a mean of 20.4 years (range, 16.3 to 24.5 years) after enrollment. The mean NAHS was 79 (range, 35 to 100), and the mean IHS was 74 (range, 43 to 100). Three patients had required hip arthroplasty and one patient had required a pelvic osteotomy. Fourteen (26%) of the remaining hips had no hip osteoarthritis, sixteen (30%) had mild osteoarthritis (Tönnis grade 1), and twenty-four (44%) had moderate or severe osteoarthritic changes on radiographs (grade 2 or 3). Femoroacetabular impingement indicated by physical examination was associated with pain and with poorer outcomes on the IHS and the NAHS (p = 0.0004, 0.0014, and 0.0007, respectively). The Stulberg classification was significantly associated with impingement on physical examination (p = 0.0495), the NAHS (p = 0.003), and the Tönnis grade (p = 0.012). Multivariate logistic regression showed that only the Stulberg classification was significantly associated with the NAHS (p = 0.0032); the odds ratio for a Stulberg type of I or II compared with IV or V in patients with a fair or poor NAHS was 0.101 (95% confidence interval, 0.018 to 0.573). Conclusions: Pain, arthritis, and ongoing hip dysfunction are common in patients with Legg-Calvé-Perthes disease that was treated nonoperatively. Hips rated as Stulberg type III or IV more frequently had poor or fair outcomes on the IHS and NAHS (61% and 72% for type III and 77% and 60% for type IV). Patients with a lateral pillar type of B, B/C, or C frequently had pain and radiographic evidence of osteoarthritis. Clinical signs of femoroacetabular impingement were associated with pain and with lower functional scores. This prospective study establishes a modern benchmark for outcomes following nonoperative, weight-bearing treatment of Legg-Calvé-Perthes disease. Level of Evidence: Prognostic Level II. See Instructions for Authors for a complete description of levels of evidence.