Osteogenic differentiation of human mesenchymal stromal cells and fibroblasts differs depending on tissue origin and replicative senescence.

The need for an autologous cell source for bone tissue engineering and medical applications has led researchers to explore multipotent mesenchymal stromal cells (MSC), which show stem cell plasticity, in various human tissues. However, MSC with different tissue origins vary in their biological properties and their capability for osteogenic differentiation. Furthermore, MSC-based therapies require large-scale ex vivo expansion, accompanied by cell type-specific replicative senescence, which affects osteogenic differentiation. To elucidate cell type-specific differences in the osteogenic differentiation potential and replicative senescence, we analysed the impact of BMP and TGF-ß signaling in adipose-derived stromal cells (ASC), fibroblasts (FB), and dental pulp stromal cells (DSC). We used inhibitors of BMP and TGF-ß signaling, such as SB431542, dorsomorphin and/or a supplemental addition of BMP-2. The expression of high-affinity binding receptors for BMP-2 and calcium deposition with alizarin red S were evaluated to assess osteogenic differentiation potential. Our study demonstrated that TGF-ß signaling inhibits osteogenic differentiation of ASC, DSC and FB in the early cell culture passages. Moreover, DSC had the best osteogenic differentiation potential and an activation of BMP signaling with BMP-2 could further enhance this capacity. This phenomenon is likely due to an increased expression of activin receptor-like kinase-3 and -6. However, in DSC with replicative senescence (in cell culture passage 10), osteogenic differentiation sharply decreased, and the simultaneous use of BMP-2 and SB431542 did not result in further improvement of this process. In comparison, ASC retain a similar osteogenic differentiation potential regardless of whether they were in the early (cell culture passage 3) or later (cell culture passage 10) stages. Our study elucidated that ASC, DSC, and FB vary functionally in their osteogenic differentiation, depending on their tissue origin and replicative senescence. Therefore, our study provides important insights for cell-based therapies to optimize prospective bone tissue engineering strategies.

Hypoxia-selective allosteric destabilization of activin receptor-like kinases: A potential therapeutic avenue for prophylaxis of heterotopic ossification.

Heterotopic ossification (HO), the pathological extraskeletal formation of bone, can arise from blast injuries, severe burns, orthopedic procedures and gain-of-function mutations in a component of the bone morphogenetic protein (BMP) signaling pathway, the ACVR1/ALK2 receptor serine-threonine (protein) kinase, causative of Fibrodysplasia Ossificans Progressiva (FOP). All three ALKs (-2, -3, -6) that play roles in bone morphogenesis contribute to trauma-induced HO, hence are well-validated pharmacological targets. That said, development of inhibitors, typically competitors of ATP binding, is inherently difficult due to the conserved nature of the active site of the 500+ human protein kinases. Since these enzymes are regulated via inherent plasticity, pharmacological chaperone-like drugs binding to another (allosteric) site could hypothetically modulate kinase conformation and activity. To test for such a mechanism, a surface pocket of ALK2 kinase formed largely by a key allosteric substructure was targeted by supercomputer docking of drug-like compounds from a virtual library. Subsequently, the effects of docked hits were further screened in vitro with purified recombinant kinase protein. A family of compounds with terminal hydrogen-bonding acceptor groups was identified that significantly destabilized the protein, inhibiting activity. Destabilization was pH-dependent, putatively mediated by ionization of a histidine within the allosteric substructure with decreasing pH. In vivo, nonnative proteins are degraded by proteolysis in the proteasome complex, or cellular trashcan, allowing for the emergence of therapeutics that inhibit through degradation of over-active proteins implicated in the pathology of diseases and disorders. Because HO is triggered by soft-tissue trauma and ensuing hypoxia, dependency of ALK destabilization on hypoxic pH imparts selective efficacy on the allosteric inhibitors, providing potential for safe prophylactic use.

Investigational anabolic therapies for osteoporosis.

IMPORTANCE OF THE FIELD: Anabolic therapy, or stimulating the function of bone-forming osteoblasts, is the preferred pharmacological intervention for osteoporosis. AREAS COVERED IN THIS REVIEW: We reviewed bone anabolic agents currently under active investigation. The bone anabolic potential of IGF-I and parathyroid hormone-related protein is discussed in the light of animal data and human studies. We also discuss the use of antagonists of the calcium-sensing receptor (calcilytics) as orally administered small molecules capable of transiently elevating serum parathyroid hormone (PTH). Further, we reviewed novel anabolic agents targeting members of the wingless tail (Wnt) signaling family that regulate bone formation including DKK-1, sclerostin, Thp1, and glycogen synthase kinase 3beta. We have also followed up on the promise shown by beta-blockers in modulating the activity of sympathetic nervous system, thus affecting bone anabolism. We give critical consideration to neutralizing the activity of activin A, a negative regulator of bone mass by soluble activin receptor IIA, as a strategy to promote bone formation. WHAT THE READER WILL GAIN: Update on various strategies to promote osteoblast function currently under evaluation. TAKE HOME MESSAGE: In spite of favorable results in experimental models, none of these strategies has yet achieved the ultimate goal of providing an alternative to injectable PTH, the sole anabolic therapy in clinical use.