The factors contributing to heterotopic ossification, the formation of bone in abnormal soft-tissue locations, are beginning to emerge, but little is known about microenvironmental conditions promoting this often devastating disease. Using a murine model in which endochondral bone formation is triggered in muscle by bone morphogenetic protein 2 (BMP2), we studied changes near the site of injection of BMP2-expressing cells. As early as 24 hours later, brown adipocytes began accumulating in the lesional area. These cells stained positively for pimonidazole and therefore generated hypoxic stress within the target tissue, a prerequisite for the differentiation of stem cells to chondrocytes and subsequent heterotopic bone formation. We propose that aberrant expression of BMPs in soft tissue stimulates production of brown adipocytes, which drive the early steps of heterotopic endochondral ossification by lowering oxygen tension in adjacent tissue, creating the correct environment for chondrogenesis. Results in misty gray lean mutant mice not producing brown fat suggest that white adipocytes convert into fat-oxidizing cells when brown adipocytes are unavailable, providing a compensatory mechanism for generation of a hypoxic microenvironment. Manipulation of the transcriptional control of adipocyte fate in local softtissue environments may offer a means to prevent or treat development of bone in extraskeletal sites. (Am

Pregnancy has a profound effect on the human body, particularly the musculoskeletal system. Hormonal changes cause ligamentous joint laxity, weight gain, and a shift in the center of gravity that leads to lumbar spine hyperlordosis and anterior tilting of the pelvis. In addition, vascular changes may lead to compromised metabolic supply in the low back. The most common musculoskeletal complaints in pregnancy are low back pain and/or pelvic girdle pain. They can be diagnosed and differentiated from each other by history taking, clinical examination, provocative test maneuvers, and imaging. Management ranges from conservative and pharmacologic measures to surgical treatment. Depending on the situation, and given the unique challenges pregnancy places on the human body and the special consideration that must be given to the fetus, an orthopaedic surgeon and the obstetrician may have to develop a plan of care together regarding labor and delivery or when surgical interventions are indicated.

Recombinant adenoviral vectors have potential for the treatment of a variety of musculoskeletal defects and such gene therapy systems have been a recent research focus in orthopedic surgery. In studies reported here, two different adenovirus vectors have been compared for their ability to transduce human bone marrow mesenchymal stem cells (hBM-MSCs) and elicit bone formation in vivo. Vectors consisted either of standard adenovirus type 5 (Ad5) vector or a chimeric adenovirus type 5 vector that contains an adenovirus type 35 fiber (Ad5F35), which has been recently demonstrated to bestow a different cellular tropism, and a complete cDNA encoding human bone morphogenetic 2 (BMP2). Studies were also conducted to compare the transduction efficiency of these vectors using enhanced green fluorescent protein (GFP). hBM-MSCs transduced with Ad5F35 vectors had higher levels of transgene expression than those transduced with Ad5 vectors. The results also demonstrate that hBM-MSCs lack the coxsackie-adenovirus receptor (CAR), which is responsible for cellular adsorption of Ad5. Therefore, the data suggest that Ad5 virus adsorption to hBM-MSCs is inefficient. Ad5BMP2- or Ad5F35BMP2-transduced hBM-MSCs were also compared in an in vivo heterotopic bone formation assay. Mineralized bone was radiologically identified only in muscle that received the Ad5F35BMP2 transduced hBM-MSCs. In summary, Ad5F35BMP2 can efficiently transduce hBM-MSCs leading to enhanced bone formation in vivo.

Osteoblasts are continually recruited from stem cell pools to maintain bone. Although their immediate precursor is a plasticadherent mesenchymal stem cell able to generate tissues other than bone, increasing evidence suggests the existence of a more primitive cell that can differentiate to both hematopoietic and mesenchymal cells. We show here that the ''side population'' (SP) of marrow stem cells, defined by their ability to rapidly expel a DNA-binding dye and to regenerate the hematopoietic compartment, can differentiate to osteoblasts through a mesenchymal intermediate. When transplanted into lethally irradiated mice, single gene-marked murine SP cells reconstituted depleted osteoprogenitor pools, such that a large proportion of the osteogenic cells in the epiphysis of long bone carried the donor SP cell marker. These findings suggest that the developmental capacity of SP cells is not restricted to the hematopoietic lineages but extends to osteogenic differentiation. This property not only elucidates a previously unrecognized step in osteoblast development, but also has intriguing implications for the use of SP cells in clinical orthopedics and stem cell-based disorders of bone. Skeletal bone is unique among human tissues. It is continuously remodeled throughout life in a process that requires the recruitment and proliferation of stem cells with the capacity to differentiate to functional osteoblasts, which then deposit and mineralize extracellular bone matrix (1, 2). The identification of an osteogenic stem cell with competency for both self-renewal and robust differentiation to bone-forming osteoblasts has been elusive. Several studies have documented the ability of cells in whole bone marrow to form osteoblasts in vitro (3) and in vivo (4). Pittenger et al. (3) isolated mesenchymal stem cells (MSCs) that were able to differentiate to chondrocytes, adipocytes, and osteoblasts in culture, whereas the results of serial transplantation of single bone marrow-derived stem cells can be interpreted to suggest the existence of rare long-term repopulating cells that can regenerate not only the entire hematopoietic system, but also several different mesenchymal lineages (5, 6).Our efforts to identify an osteogenic stem cell have focused on a side population (SP) of bone marrow cells that display strong hematopoietic reconstituting activity, as measured by competitive repopulation assays (7,8). These so-called SP cells, which can be identified by their unique capacity to efflux fluorescent DNA-binding dye (7,8), also have a limited capacity to differentiate in vivo to skeletal myocytes (9) as well as vascular endothelial cells (10), suggesting multilineage potential. To test the candidacy of these adult stem cells as progenitors of the osteoblast lineage, we tracked the fate of gene-marked SP both in vitro and in vivo. Donor-derived mesenchymal progenitors differentiated to osteoblasts in clonogenic medium, and immunostaining of long-bone sections after transplantation of SP cells into lethally irradiated mice demonst...

SUMMARY The fate of disseminated tumor cells is largely determined by microenvironment (ME) niche. The osteogenic niche promotes cancer cell proliferation and bone metastasis progression. We investigated the underlying mechanisms using pre-clinical models and analyses of clinical data. We discovered that the osteogenic niche serves as a calcium (Ca) reservoir for cancer cells through gap junction (GJs). Cancer cells cannot efficiently absorb Ca from ME, but depend on osteogenic cells to increase intracellular Ca concentration. The Ca signaling, together with previously identified mTOR signaling, promotes bone metastasis progression. Interestingly, effective inhibition of these pathways can be achieved by danusertib, or combination of everolimus and arsenic trioxide, which provide possibilities of eliminating bone micrometastases (BMM) using clinically established drugs.

Background Heterotopic ossification (HO) is the process of bone formation at a nonskeletal site. Recently, we showed that the earliest steps occur in sensory nerves. We now extend these studies by identifying unique osteogenic progenitors within the endoneurial compartment of sensory nerves. Questions/purposes We asked: (1) What is the nature of the osteoprogenitor in the endoneurium of peripheral nerves? (2) How do osteoprogenitors travel from the nerve to the site of new bone formation? Methods HO was induced by intramuscular injection of Ad5BMP-2-transduced cells in mice. Osteoprogenitors were identified through immunohistochemistry and then quantified and further characterized by fluorescence-activated cell sorting and immunocytochemistry. The kinetics of the appearance of markers of extravasation was determined by quantitative reverse transcription-polymerase chain reaction. In each experiment mice were injected with bone morphogenetic protein-2 (BMP-2)-producing cells (experimental) or with cells transduced with empty vector or, in some cases, a group receiving no injection (control). Results Induction of HO leads to the expression, within 24 hours, of osteoblast-specific transcription factors in cells in the endoneurium followed by their coordinate disappearance from the nerve at 48 hours. They reappear in 123Clin Orthop Relat Res (2015) 473:2790-2806 DOI 10.1007 Clinical Orthopaedics and Related Research ® A Publication of The Association of Bone and Joint Surgeons® blood also at 48 hours after induction. During vessel entrance they begin to express the tight junction molecule, claudin 5. The cells expressing both the osteoblast-specific transcription factor, osterix, as well as claudin 5, then disappear from circulation at approximately 3 to 4 days by extravasation into the site of new bone formation. These endoneurial osteoprogenitors express neural markers PDGFRa, musashi-1, and the low-affinity nerve growth factor receptor p75(NTR) as well as the endothelial marker Tie-2. In a key experiment, cells that were obtained from mice that were injected with cells transduced with an empty vector, at 2 days after injection, contained 0.83% (SD, 0.07; 95% confidence interval [CI], 0.59-1.05) cells expressing claudin 5. However, cells that were obtained from mice 2 days after injection of BMP-2-producing cells contained 4.5% cells expressing claudin 5 (SD, 0.72%; 95% CI, 2.01-6.94; p \ 0.0015). Further analysis revealed that all of the cells expressing claudin 5 were found to be positive for osteoblast-specific markers, whereas cells not expressing claudin 5 were negative for these same markers. Conclusions The findings suggest that the endoneurial progenitors are the major osteogenic precursors that are used for HO. They exit the nerve through the endoneurial vessels, flow through vessels to the site of new bone formation, and then extravasate out of the vessels into this site. Clinical Relevance The biogenesis of osteoblasts in HO is very different than expected and shows that HO is, at least in par...

Recombinant adenoviral vectors have been shown to be potential new tools for a variety of musculoskeletal defects. Much emphasis in the field of orthopedic research has been placed on developing systems for the production of bone. This study aims to determine the necessary conditions for sustained production of high levels of active bone morphogenetic protein 2 (BMP2) using a recombinant adenovirus type 5 (Ad5BMP2) capable of eliciting BMP2 synthesis upon infection and to evaluate the consequences for osteoprogenitor cells. The results indicate that high levels (144 ng/ml) of BMP2 can be produced in non-osteoprogenitor cells (A549 cell line) by this method and the resultant protein appears to be three times more biologically active than the recombinant protein. Surprisingly, similar levels of BMP2 expression could not be achieved after transduction with Ad5BMP2 of either human bone marrow stromal cells or the mouse bone marrow stromal cell line W20-17. However, human bone marrow stromal cells cultured with 1 microM dexamethasone for four days, or further stimulated to become osteoblast-like cells with 50 microg/ml ascorbic acid, produced high levels of BMP2 upon Ad5BMP2 infection as compared to the undifferentiated cells. The increased production of BMP2 in adenovirus transduced cells following exposure to 1 microM dexamethasone was reduced if the cells were not given 50 microg/ml ascorbic acid. When bone marrow stromal cells were allowed to become confluent in culture prior to differentiation, BMP2 production in response to Ad5BMP2 infection was lost entirely. Furthermore, the increase in BMP2 synthesis seen during differentiation was greatly decreased when Ad5BMP2 was administered prior to dexamethasone treatment. In short, the efficiency of adenovirus mediated expression of BMP2 in bone marrow stromal cells appears to be dependent on the differentiation state of these cells.

SEMAC-VAT (2D) and MSVAT-SPACE (3D) demonstrated a consistent, marked reduction of metal artifacts for different metal implants and offered flexible image contrasts (T1, T2, PD and STIR) with high image quality. These techniques likely will improve the evaluation of postoperative patients with metal implants.