Bone is maintained by coupled activities of bone-forming osteoblasts/osteocytes and bone-resorbing osteoclasts. Alterations in this relationship can lead to pathologic bone loss such as osteoporosis. It is well known that osteogenic cells support osteoclastogenesis via production of RANKL. Interestingly, our recently identified bone marrow mesenchymal cell population—marrow adipogenic lineage precursors (MALPs) that form a multidimensional cell network in bone—was computationally demonstrated to be the most interactive with monocyte-macrophage lineage cells through high and specific expression of several osteoclast regulatory factors, including RANKL. Using an adipocyte-specific Adipoq-Cre to label MALPs, we demonstrated that mice with RANKL deficiency in MALPs have a drastic increase in trabecular bone mass in long bones and vertebrae starting from 1 month of age, while their cortical bone appears normal. This phenotype was accompanied by diminished osteoclast number and attenuated bone formation at the trabecular bone surface. Reduced RANKL signaling in calvarial MALPs abolished osteolytic lesions after LPS injections. Furthermore, in ovariectomized mice, elevated bone resorption was partially attenuated by RANKL deficiency in MALPs. In summary, our studies identified MALPs as a critical player in controlling bone remodeling during normal bone metabolism and pathological bone loss in a RANKL-dependent fashion.

Presently, there is a high paucity of bone grafts in the United States and worldwide. Regenerating bone is of prime concern due to the current demand of bone grafts and the increasing number of diseases causing bone loss. Autogenous bone is the present gold standard of bone regeneration. However, disadvantages like donor site morbidity and its decreased availability limit its use. Even allografts and synthetic grafting materials have their own limitations. As certain specific stem cells can be directed to differentiate into an osteoblastic lineage in the presence of growth factors (GFs), it makes stem cells the ideal agents for bone regeneration. Furthermore, platelet-rich plasma (PRP), which can be easily isolated from whole blood, is often used for bone regeneration, wound healing and bone defect repair. When stem cells are combined with PRP in the presence of GFs, they are able to promote osteogenesis. This review provides in-depth knowledge regarding the use of stem cells and PRPin vitro,in vivoand their application in clinical studies in the future.

Human insulin-like growth factor-1 (IGF-1) plays important roles in development and regeneration of skeletal muscles and bones but requires daily injections or surgical implantation. Current clinical IGF-1 lacks e-peptide and is glycosylated, reducing functional efficacy. In this study, codon-optimized Pro-IGF-1 with e-peptide (fused to GM1 receptor binding protein CTB or cell penetrating peptide PTD) was expressed in lettuce chloroplasts to facilitate oral delivery. Pro-IGF-1 was expressed at high levels in the absence of the antibiotic resistance gene in lettuce chloroplasts and was maintained in subsequent generations. In lyophilized plant cells, Pro-IGF-1 maintained folding, assembly, stability and functionality up to 31 months, when stored at ambient temperature. CTB-Pro-IGF-1 stimulated proliferation of human oral keratinocytes, gingiva-derived mesenchymal stromal cells and mouse osteoblasts in a dose-dependent manner and promoted osteoblast differentiation through upregulation of ALP, OSX and RUNX2 genes. Mice orally gavaged with the lyophilized plant cells significantly increased IGF-1 levels in sera, skeletal muscles and was stable for several hours. When bioencapsulated CTB-Pro-IGF-1 was gavaged to femoral fractured diabetic mice, bone regeneration was significantly promoted with increase in bone volume, density and area. This novel delivery system should increase affordability and patient compliance, especially for treatment of musculoskeletal diseases.

The primary cilium is a nonmotile microtubule-based organelle in most vertebrate cell types. The primary cilium plays a critical role in tissue development and homeostasis by sensing and transducing various signaling pathways. Ciliary proteins such as intraflagellar transport (IFT) proteins as well as ciliary motor proteins, kinesin and dynein, comprise a bidirectional intraflagellar transport system needed for cilia formation and function. Mutations in ciliary proteins that lead to loss or dysfunction of primary cilia cause ciliopathies such as Jeune syndrome and Ellis–van Creveld syndrome and cause abnormalities in tooth development. These diseases exhibit severe skeletal and craniofacial dysplasia, highlighting the significance of primary cilia in skeletal development. Cilia are necessary for the propagation of hedgehog, transforming growth factor β, platelet-derived growth factor, and fibroblast growth factor signaling during osteogenesis and chondrogenesis. Ablation of ciliary proteins such as IFT80 or IFT20 blocks cilia formation, which inhibits osteoblast differentiation, osteoblast polarity, and alignment and reduces bone formation. Similarly, cilia facilitate chondrocyte differentiation and production of a cartilage matrix. Cilia also play a key role in mechanosensing and are needed for increased bone formation in response to mechanical forces.

Repair and regeneration of inflammation-induced bone loss remains a clinical challenge. LL37, an antimicrobial peptide, plays critical roles in cell migration, cytokine production, apoptosis, and angiogenesis. Migration of stem cells to the affected site and promotion of vascularization are essential for tissue engineering therapy, including bone regeneration. However, it is largely unknown whether LL37 affects mesenchymal stem cell (MSC) behavior and bone morphogenetic protein 2 (BMP2)-mediated bone repair during the bone pathologic remodeling process. By performing in vitro and in vivo studies with MSCs and a lipopolysaccharide (LPS)-induced mouse calvarial osteolytic bone defect model, we found that LL37 significantly promotes cell differentiation, migration, and proliferation in both unmodified MSCs and BMP2 gene-modified MSCs. Additionally, LL37 inhibited LPS-induced osteoclast formation and bacterial activity in vitro. Furthermore, the combination of LL37 and BMP2 markedly promoted MSC-mediated angiogenesis and bone repair and regeneration in LPS-induced osteolytic defects in mouse calvaria. These findings demonstrate for the first time that LL37 can be a potential candidate drug for promoting osteogenesis and for inhibiting bacterial growth and osteoclastogenesis, and that the combination of BMP2 and LL37 is ideal for MSC-mediated bone regeneration, especially for inflammation-induced bone loss.

Osteoclasts are bone‐resorbing cells differentiated from macrophage/monocyte precursors in response to macrophage colony‐stimulating factor (M‐CSF) and receptor activator of NF‐κB ligand (RANKL). In vitro models are principally based on primary bone marrow macrophages (BMMs), but RAW 264.7 cells are frequently used because they are widely available, easy to culture, and more amenable to genetic manipulation than primary cells. Increasing evidence, however, has shown that the vastly different origins of these two cell types may have important effects on cell behavior. In particular, M‐CSF is a prerequisite for the differentiation of BMMs, by promoting survival and proliferation and priming the cells for RANKL induction. RAW 264.7 cells readily form osteoclasts in the presence of RANKL, but M‐CSF is not required. Based on these key differences, we sought to understand their functional implications and how it might affect osteoclast differentiation and related signaling pathways. Using a robust and high‐throughput proteomics strategy, we quantified the global protein changes in osteoclasts derived from BMMs and RAW 264.7 cells at 1, 3, and 5 days of differentiation. Data are available via ProteomeXchange with the identifier PXD009610. Correlation analysis of the proteomes demonstrated low concordance between the two cell types (R² ≈ 0.13). Bioinformatics analysis indicate that RANKL‐dependent signaling was intact in RAW 264.7 cells, but biological processes known to be dependent on M‐CSF were significantly different, including cell cycle control, cytoskeletal organization, and apoptosis. RAW 264.7 cells exhibited constitutive activation of Erk and Akt that was dependent on the activity of Abelson tyrosine kinase, and the timing of Erk and Akt activation was significantly different between BMMs and RAW 264.7 cells. Our findings provide the first evidence for major discrepancies between BMMs and RAW 264.7 cells, indicating that careful consideration is needed when using the RAW 264.7 cell line for studying M‐CSF‐dependent signaling and functions. © 2018 American Society for Bone and Mineral Research. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.