Elucidating the molecular mechanisms that regulate human stromal (mesenchymal) stem cell (hMSC) differentiation into osteogenic lineage is important for the development of anabolic therapies for treatment of osteoporosis. MicroRNAs (miRNAs) are short, noncoding RNAs that act as key regulators of diverse biological processes by mediating translational repression or mRNA degradation of their target genes. Here, we show that miRNA-138 (miR-138) modulates osteogenic differentiation of hMSCs. miRNA array profiling and further validation by quantitative RT-PCR (qRT-PCR) revealed that miR-138 was down-regulated during osteoblast differentiation of hMSCs. Overexpression of miR-138 inhibited osteoblast differentiation of hMSCs in vitro, whereas inhibition of miR-138 function by antimiR-138 promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Furthermore, overexpression of miR-138 reduced ectopic bone formation in vivo by 85%, and conversely, in vivo bone formation was enhanced by 60% when miR-138 was antagonized. Target prediction analysis and experimental validation by luciferase 3′ UTR reporter assay confirmed focal adhesion kinase, a kinase playing a central role in promoting osteoblast differentiation, as a bona fide target of miR-138. We show that miR-138 attenuates bone formation in vivo, at least in part by inhibiting the focal adhesion kinase signaling pathway. Our findings suggest that pharmacological inhibition of miR-138 by antimiR-138 could represent a therapeutic strategy for enhancing bone formation in vivo.

• Introduction • Bone fracture healing and healing problems • Biomaterial scaffolds and tissue engineering in bone formation ‐ Bone tissue engineering ‐ Biomaterial scaffolds ‐ Synthetic scaffolds ‐ Micro‐ and nanostructural properties of scaffolds ‐ Conclusion • Mesenchymal stem cells and osteogenesis ‐ Bone tissue ‐ Origin of osteoblasts ‐ Isolation and characterization of bone marrow derived MSC ‐ In vitro differentiation of MSC into osteoblast lineage cells ‐ In vivo differentiation of MSC into bone ‐ Factors and pathways controlling osteoblast differentiation of hMSC ‐ Defining the relationship between osteoblast and adipocyte differentiation from MSC ‐ MSC and sex hormones ‐ Effect of aging on osteoblastogenesis ‐ Conclusion • Embryonic, foetal and adult stem cells in osteogenesis ‐ Cell‐based therapies for bone ‐ Specific features of bone cells needed to be advantageous for clinical use ‐ Development of therapeutic biological agents ‐ Clinical application concerns ‐ Conclusion • Platelet‐rich plasma (PRP), growth factors and osteogenesis ‐ PRP effects in vitro on the cells involved in bone repair ‐ PRP effects on osteoblasts ‐ PRP effects on osteoclasts ‐ PRP effects on endothelial cells ‐ PRP effects in vivo on experimental animals ‐ The clinical use of PRP for bone repair ‐ Non‐union ‐ Distraction osteogenesis ‐ Spinal fusion ‐ Foot and ankle surgery ‐ Total knee arthroplasty ‐ Odontostomatology and maxillofacial surgery ‐ Conclusion • Molecular control of osteogenesis ‐ TGF‐β signalling ‐ FGF signalling ‐ IGF signalling ‐ PDGF signalling ‐ MAPK signalling pathway ‐ Wnt signalling pathway ‐ Hedgehog signalling ‐ Notch signalling ‐ Ephrin signalling ‐ Transcription factors regulating osteoblast differentiation ‐ Conclusion • Summary This invited review covers research areas of central importance for orthopaedic and maxillofacial bone tissue repair, including normal fracture healing and healing problems, biomaterial scaffolds for tissue engineering, mesenchymal and foetal stem cells, effects of sex steroids on mesenchymal stem cells, use of platelet‐rich plasma for tissue repair, osteogenesis and its molecular markers. A variety of cells in addition to stem cells, as well as advances in materials science to meet specific requirements for bone and soft tissue regeneration by addition of bioactive molecules, are discussed.

Dlk-1/Pref-1 was identified as a novel regulator of human skeletal stem cell differentiation. Dlk1/Pref-1 is expressed in bone and cultured osteoblasts, and its constitutive overexpression led to inhibition of osteoblast and adipocyte differentiation of human marrow stromal cells. Introduction: Molecular control of human mesenchymal stem cell (hMSC) differentiation into osteoblasts and adipocytes is not known. In this study, we examined the role of delta-like 1/preadipocyte factor-1 (Dlk1/Pref-1) in regulating the differentiation of hMSCs. Materials and Methods: As a model for hMSCs, we have stably transduced telomerase-immortalized hMSC (hMSC-TERT) with the full length of human Dlk1/Pref-1 cDNA and tested its effect on hMSC growth and differentiation into osteoblasts or adipocytes as assessed by cytochemical staining, FACS analysis, and real time PCR. Ex vivo calvaria organ cultures assay was used to confirm the in vitro effect of Dlk/Pref-1 on bone formation. Results: Dlk1/Pref-1 was found to be expressed in fetal and adult bone, hMSCs, and some osteoblastic cell lines. A retroviral vector containing the human Dlk1/Pref-1 cDNA was used to create a cell line (hMSC-dlk1) expressing high levels of Dlk1/Pref-1 protein. Overexpression of Dlk1/Pref-1 did not affect the proliferation rate of hMSC, but the ability to form mature adipocytes, mineralized matrix in vitro, and new bone formation in neonatal murine calvariae organ cultures was reduced. These effects were associated with inhibition of gene expression markers of late stages of adipocyte (adipocyte fatty acid-binding protein [aP2], peroxisome proliferator-activated receptor-gamma2 [PPARγ2], and adiponectin [APM1]) and osteoblast differentiation (alkaline phosphatase [ALP], collagen type I [Col1], and osteocalcin [OC]). Lineage commitment markers for adipocytes (adipocyte determination and differentiation factor −1 [ADD1]) and osteoblasts (core binding factor/runt-related binding factor 2 [Cbfa1/Runx2]) were not affected. Conclusion: During hMSC differentiation, Dlk1/Pref-1 maintains the size of the bipotential progenitor cell pool by inhibiting the formation of mature osteoblasts and adipocytes.