A novel in vitro assay to study chondrocyte-to-osteoblast transdifferentiation

Purpose Endochondral ossification, which involves transdifferentiation of chondrocytes into osteoblasts, is an important process involved in the development and postnatal growth of most vertebrate bones as well as in bone fracture healing. To study the basic molecular mechanisms of this process, a robust and easy-to-use in vitro model is desirable. Therefore, we aimed to develop a standardized in vitro assay for the transdifferentiation of chondrogenic cells towards the osteogenic lineage. Methods Murine chondrogenic ATDC5 cells were differentiated into the chondrogenic lineage for seven days and subsequently differentiated towards the osteogenic direction. Gene expression analysis of pluripotency, as well as chondrogenic and osteogenic markers, cell–matrix staining, and immunofluorescent staining, were performed to assess the differentiation. In addition, the effects of Wnt3a and lipopolysaccharides (LPS) on the transdifferentiation were tested by their addition to the osteogenic differentiation medium. Results Following osteogenic differentiation, chondrogenically pe-differentiated cells displayed the expression of pluripotency and osteogenic marker genes as well as alkaline phosphatase activity and a mineralized matrix. Co-expression of Col2a1 and Col1a1 after one day of osteogenic differentiation indicated that osteogenic cells had differentiated from chondrogenic cells. Wnt3a increased and LPS decreased transdifferentiation towards the osteogenic lineage. Conclusion We successfully established a rapid, standardized in vitro assay for the transdifferentiation of chondrogenic cells into osteogenic cells, which is suitable for testing the effects of different compounds on this cellular process.

Osteogenic Potential of Mesenchymal Stem Cells from Adipose Tissue, Bone Marrow and Hair Follicle Outer Root Sheath in a 3D Crosslinked Gelatin-Based Hydrogel

Bone transplantation is regarded as the preferred therapy to treat a variety of bone defects. Autologous bone tissue is often lacking at the source, and the mesenchymal stem cells (MSCs) responsible for bone repair mechanisms are extracted by invasive procedures. This study explores the potential of autologous mesenchymal stem cells derived from the hair follicle outer root sheath (MSCORS). We demonstrated that MSCORS have a remarkable capacity to differentiate in vitro towards the osteogenic lineage. Indeed, when combined with a novel gelatin-based hydrogel called Osteogel, they provided additional osteoinductive cues in vitro that may pave the way for future application in bone regeneration. MSCORS were also compared to MSCs from adipose tissue (ADMSC) and bone marrow (BMMSC) in a 3D Osteogel model. We analyzed gel plasticity, cell phenotype, cell viability, and differentiation capacity towards the osteogenic lineage by measuring alkaline phosphatase (ALP) activity, calcium deposition, and specific gene expression. The novel injectable hydrogel filled an irregularly shaped lesion in a porcine wound model displaying high plasticity. MSCORS in Osteogel showed a higher osteo-commitment in terms of calcium deposition and expression dynamics of OCN, BMP2, and PPARG when compared to ADMSC and BMMSC, whilst displaying comparable cell viability and ALP activity. In conclusion, autologous MSCORS combined with our novel gelatin-based hydrogel displayed a high capacity for differentiation towards the osteogenic lineage and are acquired by non-invasive procedures, therefore qualifying as a suitable and expandable novel approach in the field of bone regeneration therapy.

Epigenetic Control of Osteogenic Lineage Commitment

Within the eukaryotic nucleus the genomic DNA is organized into chromatin by stably interacting with the histone proteins as well as with several other nuclear components including non-histone proteins and non-coding RNAs. Together these interactions distribute the genetic material into chromatin subdomains which can exhibit higher and lower compaction levels. This organization contributes to differentially control the access to genomic sequences encoding key regulatory genetic information. In this context, epigenetic mechanisms play a critical role in the regulation of gene expression as they modify the degree of chromatin compaction to facilitate both activation and repression of transcription. Among the most studied epigenetic mechanisms we find the methylation of DNA, ATP-dependent chromatin remodeling, and enzyme-mediated deposition and elimination of post-translational modifications at histone and non-histone proteins. In this mini review, we discuss evidence that supports the role of these epigenetic mechanisms during transcriptional control of osteoblast-related genes. Special attention is dedicated to mechanisms of epigenetic control operating at the Runx2 and Sp7 genes coding for the two principal master regulators of the osteogenic lineage during mesenchymal stem cell commitment.

Porous Silk Fibroin/Cellulose Hydrogels for Bone Tissue Engineering via a Novel Combined Process Based on Sequential Regeneration and Porogen Leaching

Scaffolds used for bone tissue engineering need to have a variety of features to accommodate bone cells. The scaffold should mimic natural bone, it should have appropriate mechanical strength, support cell differentiation to the osteogenic lineage, and offer adequate porosity to allow vascularization and bone in-growth. In this work, we aim at developing a new process to fabricate such materials by creating a porous composite material made of silk fibroin and cellulose as a suitable scaffold of bone tissue engineering. Silk fibroin and cellulose are both dissolved together in N,N-dimethylacetamide/LiCl and molded to a porous structure using NaCl powder. The hydrogels are prepared by a sequential regeneration process: cellulose is solidified by water vapor treatment, while the remaining silk fibroin in the hydrogel is insolubilized by methanol, which leads to a cellulose framework structure embedded in a silk fibroin matrix. Finally, the hydrogels are soaked in water to dissolve the NaCl for making a porous structure. The cellulose composition results in improving the mechanical properties for the hydrogels in comparison to the silk fibroin control material. The pore size and porosity are estimated at around 350 µm and 70%, respectively. The hydrogels support the differentiation of MC3T3 cells to osteoblasts and are expected to be a good scaffold for bone tissue engineering.

Osteogenic differentiation potential of porcine bone marrow mesenchymal stem cell subpopulations selected in different basal media

ABSTRACTMultipotent porcine mesenchymal stem cells (pMSC) are invaluable for research and therapeutic use in regenerative medicine. Media used for derivation and expansion of pMSC may play an important role for the selection of MSC subpopulation at an early stage and thereby, the specific basal medium may also affect differentiation potential of these cells. The present study was undertaken to evaluate the effects of αMEM, aDMEM, M199, αMEM/M199, aDMEM/M199 and αMEM/aDMEM media on (1) porcine bone marrow MSC derivation; (2) expression of number of osteogenic markers (ALP, COL1A1, SPP1 and BGLAP) at 5th and 10th passage in pMSC before differentiation; and (3) differentiation of pMSC (at 5th passage) to osteogenic lineage. Morphological changes and matrix formation in osteogenic cells were evaluated by microscopic examination. Calcium deposits in osteocytes were confirmed by Alizarin Red S staining. Based on expression of different markers, it was evident that selection of bone marrow pMSC subpopulations was independent of basal media used. However, the differentiation of those pMSCs, specifically to osteogenic lineage, was dependent on the medium used for expansion of pMSC at the pre-differentiation stage. We demonstrated here that the pMSC grown in combined αMEM/aDMEM (1:1) medium expressed number of osteogenic markers and these pMSC underwent osteogenic differentiation most efficiently, in comparison to porcine mesenchymal stem cells grown in other media. In conclusion, osteogenic differentiation potential of pMSC maintained in αMEM/aDMEM medium was observed significantly higher compared to cells cultivated in other media and therefore, the combined medium αMEM/aDMEM (1:1) may preferentially be used for expansion of pMSC, if needed for osteogenic differentiation.