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.

The need for a suitable scaffolding material for load bearing bone tissue engineering still has yet to be met satisfactorily. In this study, Portland cement and Portland cement/metakaolin (MK) blends were processed to render them biologically and mechanically suitable for such application. Portland cement was mixed with MK at different ratios. The slurries were hydrated under atmospheric (noncarbonated samples) and high-CO₂ conditions (carbonated samples). The mechanical properties were characterized via compressive tests. The bioactivity was analyzed in a simulated body fluid solution. Scanning electron microscopy and energy dispersive spectroscopy were used to evaluate sample morphology and chemistry. The cytocompatibility (direct contact assay, MTT test, and alkaline phosphatase activity) was tested using human osteoblast-like cells. Cell responses were observed via conventional and electron microscopy. The results showed that the implementation of MK did not significantly influence the mechanical properties. All the samples evidenced bioactive behavior. Cell experiments confirmed a highly cytotoxic response to the noncarbonated specimens. The introduction of MK as well as the CO₂ pretreatment significantly improved the cytocompatibility of the specimens. These results show that properly processed Portland cement and Portland cement/MK blends could present suitable properties for the development of load-bearing scaffolding structures in bone tissue-engineering applications.

We studied the potential applications of Portland cement and Portland cementMetakaolin blends as scaffolding materials for load bearing bone tissue engineering. Cementitious pastes were prepared by mixing Portland cement and Metakaolin at different ratios (100:0, 85:15), and hydrated under a concentrated CO 2 atmosphere (carbonated pastes). Pastes fabricated similarly, but hydrated under normal atmospheric conditions were used for comparison (non-carbonated pastes). Compressive tests were run to evaluate the mechanical properties of the pastes. The bioactivity of the samples was tested in a simulated body fluid (SBF) solution for 1 and 4 days. Sample morphology and chemistry were characterized via scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS), respectively. The cytocompatibility was studied using human osteosarcoma (HOS) cell cultures and the direct contact assay. Mechanical characterization did not show significant differences in the compressive strength of the blends compared to pure cement controls. The bioactivity test revealed that the pastes induced surface precipitation of calcium phosphate (CaP) when exposed to the SBF solution (as confirmed by SEM and EDS). Non-carbonated pastes induced early CaP precipitation. Cytocompatibility experiments showed that the carbonated blends allowed adequate cell growth. Non-carbonated blends presented a highly cytotoxic behavior. The introduction of Metakaolin did not affect the cytocompatibility of the samples. These results show that Portland cement and Portland cement-Metakaolin blends could present suitable characteristics for applications as scaffolding materials in load bearing bone tissue engineering.