Real-time reverse transcription PCR (RT-qPCR) relies on a housekeeping or normalizer gene whose expression remains constant throughout the experiment. RT-qPCR is commonly used for characterization of human bone marrow mesenchymal stem cells (hBMSCs). However, to the best of our knowledge, there are no studies validating the expression stability of the genes used as normalizers during hBMSCs differentiation. This work aimed to study the stability of the housekeeping genes b-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal protein L13A (RPL13A) during the osteogenic differentiation of hBMSCs. Their stability was evaluated via RT-qPCR in 14 and 20 day differentiation assays to the osteogenic lineage. Different normalization strategies were evaluated to quantify the osteogenic markers collagen type I, bone sialoprotein and osteonectin. Cell differentiation was confirmed via alizarin red staining. The results demonstrated up-regulation of b-actin with maximum fold changes (MFC) of 4.38. GAPDH and RPL13A were not regulated by osteogenic media after 14 days and presented average fold changes lower than 2 in 20 day cultures. RPL13A (MFC \ 2) had a greater stability when normalizing as a function of culture time compared with GAPDH (MFC B 2.2), which resulted in expression patterns of the osteogenic markers more consistent with the observed differentiation process. The results suggest that b-actin regulation could be associated with the morphological changes characteristic of hBMSCs osteogenic differentiation, and provide evidence for the superior performance of RPL13A as a normalizer gene in osteogenic differentiation studies of hBMSCs. This work highlights the importance of validating the normalizer genes used for stem cells characterization via RT-qPCR.

Modified Portland cement porous scaffolds with suitable characteristics for load-bearing bone tissue engineering applications were manufactured by combining the particulate leaching and foaming methods. Non-crosslinked polydimethylsiloxane was evaluated as a potential reinforcing material. The scaffolds presented average porosities between 70 and 80% with mean pore sizes ranging from 300 μm up to 5.0 mm. Non-reinforced scaffolds presented compressive strengths and elastic modulus values of 2.6 and 245 MPa, respectively, whereas reinforced scaffolds exhibited 4.2 and 443 MPa, respectively, an increase of ∼62 and 80%. Portland cement scaffolds supported human osteoblast-like cell adhesion, spreading, and propagation (t = 1-28 days). Cell metabolism and alkaline phosphatase activity were found to be enhanced at longer culture intervals (t ≥ 14 days). These results suggest the possibility of obtaining strong and biocompatible scaffolds for bone repair applications from inexpensive, yet technologically advanced materials such as Portland cement.

Purpose Legg-Calvé-Perthes disease is an idiopathic avascular necrosis of the femoral head. Although many surgical approaches to treat the late presentation of this pathology have been proposed, there are few reports about the early results of the double osteotomy procedure (femoral varus osteotomy combined with Salter innominate osteotomy). The purpose of this study was to describe the early results obtained with the double osteotomy in patients with late presentation of Legg-Calvé-Perthes disease. Methods Cross-sectional evaluation of ten patients intervened with double osteotomy. There were seven males and three females with a mean age of 9.2 ± 1.7 years [standard deviation (SD)]. The average post-surgical time of evaluation was of 46.5 ± 26.2 months. Results Of the ten evaluated patients, four had a Catterall III and six had a Catterall IV disease. According to Herring classification, three patients were Herring B and seven were Herring C. The epiphyseal extrusion average before and after the surgical procedure was 19.3 ± 12.4 and 12.1 ± 14.9%, respectively. In accordance with the Ratliff classification and Lloyd Roberts radiological results, the following were the postoperative clinical results: four good, five fair and one poor. Based on the Stulberg classification, there was one patient in class I, five in class II, three in class III and one in class IV. Conclusion The surgical treatment for late Perthes disease with the best expected outcome is still a challenge. According to the resultsreported here, the double osteotomy could be considered as an alternative to treat this entity.