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We previously identified transient brown adipocyte-like cells associated with heterotopic ossification (HO). These ancillary cells support new vessel synthesis essential to bone formation. Recent studies have shown that the M2 macrophage contributes to tissue regeneration in a similar way. To further define the phenotype of these brown adipocyte-like cells they were isolated and characterized by single-cell RNAseq (scRNAseq). Analysis of the transcriptome and the presence of surface markers specific for macrophages suggest that these cells are M2 macrophages. To validate these findings, clodronate liposomes were delivered to the tissues during HO, and the results showed both a significant reduction in these macrophages as well as bone formation. These cells were isolated and shown in culture to polarize towards either M1 or M2 similar to other macrophages. To confirm that these are M2 macrophages, mice received lipopolysacheride (LPS), which induces proinflammation and M1 macrophages. The results showed a significant decrease in this specific population and bone formation, suggesting an essential role for M2 macrophages in the production of bone. To determine if these macrophages are specific to HO, we isolated these cells using fluorescence-activated cell sorting (FACS) from a bone defect model and subjected them to scRNAseq. Surprisingly, the macrophage populations overlapped between the two groups (HO-derived versus callus) suggesting that they may be essential ancillary cells for bone formation in general and not selective to HO. Of further note, their unique metabolism and lipogenic properties suggest the potential for unique cross talk between these cells and the newly forming bone.

Inflammation has a role in the pathogenesis of childhood malnutrition. We investigated the effect of malnutrition and inflammatory challenge on bone marrow composition and bone health. We studied an established murine model of moderate acute malnutrition at baseline and after acute inflammatory challenge with bacterial lipopolysaccharide (LPS), a surrogate of Gram-negative bacterial sepsis, or Leishmania donovani , the cause of visceral leishmaniasis. Both of these infections cause significant morbidity and mortality in malnourished children. Of the 2 stimuli, LPS caused more pronounced bone marrow changes that were amplified in malnourished mice. LPS challenge led to increased inflammatory cytokine expression ( Il1b , Il6 , and Tnf ), inflammasome activation, and inflammatory monocyte accumulation in the bone marrow of malnourished mice. Depletion of inflammatory monocytes in Csfr1 -LysMcre-DT malnourished mice significantly reduced the inflammasome activation and IL1-ß production after LPS challenge. The inflammatory challenge also led to increased expansion of mesenchymal stem cells (MSCs), bone marrow adiposity, and expression of genes ( Pparg , Adipoq , and Srbp1 ) associated with adipogenesis in malnourished mice. This suggests that inflammatory challenge promotes differentiation of BM MSCs toward the adipocyte lineage rather than toward bone-forming osteoblasts in the malnourished host. Concurrent with this reduced osteoblastic potential there was an increase in bone-resorbing osteoclasts, enhanced osteoclast activity, upregulation of inflammatory genes, and IL-1B involved in osteoclast differentiation and activation. The resulting weakened bone formation and increased bone resorption would contribute to the bone fragility associated with malnutrition. Lastly, we evaluated the effect of replacing lipid rich in omega-6 fatty acids (corn oil) with lipid-rich in omega-3 fatty acids (fish oil) in the nutrient-deficient diet. LPS-challenged malnourished mice that received dietary fish oil showed decreased expression of inflammatory cytokines and Rankl and reduced osteoclast differentiation and activation in the bone marrow. This work demonstrates that the negative effect of inflammatory challenge on bone marrow is amplified in the malnourished host. Increasing dietary intake of omega-3 fatty acids may be a means to reduce inflammation and improve bone health in malnourished children.

Aim. The purpose of this study is to explore clinical characteristics of patients with T2DM receiving a primary knee (TKA) or hip (THA) arthroplasty to patients without T2DM receiving a TKA or THA and patients with T2DM with no history of osteoarthritis (OA). Methods. The study included a retrospective database review of 500 consecutive primary TKA or THA identified with ICD-9 codes and 100 consecutive T2DM patients. Patients who received a TKA or THA were screened for inclusion and exclusion and divided into with or without T2DM groups. A comparison group of patients with T2DM only without arthroplasty was screened to exclude patients with a history of OA or arthroplasty. All groups were compared based on demographic and relevant comorbidity differences. OA characteristics, including OA and previous arthroplasty of the involved and contralateral joints, were compared between patients with and without T2DM receiving a TKA or THA. Finally, patients with T2DM with and without TKA or THA were compared for T2DM differences. Results. Study results found that among those receiving a primary arthroplasty, patients with T2DM were more likely to be obese and older and reported cardiovascular, urinary, dyslipidemia, and peripheral neuropathy than those with T2DM. Among the T2DM individuals, those receiving an arthroplasty surgery were older and obese and more likely to report peripheral neuropathy; however, those with T2DM with no OA were more likely to report atherosclerosis and cardiovascular disease. Within the arthroplasty subgroup of individuals with T2DM, those requiring antidiabetic medication were 4.5 times more likely to have contralateral OA or arthroplasty. Conclusions. The results of this study suggest that patients with T2DM requiring a primary arthroplasty are a unique subgroup that requires careful considerations as they are often older, have obesity, and specific comorbidities predisposing to worse postoperative outcomes than their non-T2DM arthroplasty counterparts. Therefore, clinical practice and future studies must consider strategies that would limit OA and arthroplasty management delays while accounting for comorbidities and patient characteristics.

The majority of breast cancer models for drug discovery are based on orthotopic or subcutaneous tumours. Therapeutic responses of metastases, especially microscopic metastases, are likely to differ from these tumours due to distinct cancer-microenvironment crosstalk in distant organs. Here, to recapitulate such differences, we established an ex vivo bone metastasis model, termed bone-in-culture array or BICA, by fragmenting mouse bones preloaded with breast cancer cells via intra-iliac artery injection. Cancer cells in BICA maintain features of in vivo bone micrometastases regarding the microenvironmental niche, gene expression profile, metastatic growth kinetics and therapeutic responses. Through a proof-of-principle drug screening using BICA, we found that danusertib, an inhibitor of the Aurora kinase family, preferentially inhibits bone micrometastases. In contrast, certain histone methyltransferase inhibitors stimulate metastatic outgrowth of indolent cancer cells, specifically in the bone. Thus, BICA can be used to investigate mechanisms involved in bone colonization and to rapidly test drug efficacies on bone micrometastases. The bone microenvironment may alter therapeutic responses of disseminated breast cancer cells. Here the authors establish an ex vivo bone metastasis model, termed BICA, to delineate the effects of bone microenvironment and to rapidly discover anti-metastasis drugs.

Bone morphogenetic protein 2 (BMP2)‐induced heterotopic bone formation (HBF) starts synchronously from zero upon BMP2 induction, which is advantageous for lineage tracking. The studies reported here in GLAST‐CreErt2:tdTomato red (TR)floxSTOPflox mice during BMP2‐induced HBF show 78.8 ± 11.6% of chondrocytes and 86.5 ± 1.9% of osteoblasts are TR+ after approximately 1 week. Clustering after single‐cell RNAseq resulted in nine cell types, and analysis revealed one as a highly replicating stem‐like cell (RSC). Pseudotiming suggested that the RSC transitions to a mesenchymal stem‐like cell that simultaneously expresses multiple osteoblast and chondrocyte transcripts (chondro‐osseous progenitor [COP]). RSCs and COPs were isolated using flow cytometry for unique surface markers. Isolated RSCs (GLAST‐TR+ Hmmr+ Cd200−) and COPs (GLAST‐TR+ Cd200+ Hmmr−) were injected into the muscle of mice undergoing HBF. Approximately 9% of the cells in heterotopic bone (HB) in mice receiving RSCs were GLAST‐TR+, compared with less than 0.5% of the cells in mice receiving COPs, suggesting that RSCs are many times more potent than COPs. Analysis of donor‐derived TR+ RSCs isolated from the engrafted HB showed approximately 50% were COPs and 45% were other cells, presumably mature bone cells, confirming the early nature of the RSCs. We next isolated RSCs from these mice (approximately 300) and injected them into a second animal, with similar findings upon analysis of HBF. Unlike other methodology, single cell RNAseq has the ability to detect rare cell populations such as RSCs. The fact that RSCs can be injected into mice and differentiate suggests their potential utility for tissue regeneration. Blue arrows show the trajectory of heterotopic bone formation after bone morphogenetic protein 2 induction. The replicating stem‐like cell (RSC) is an epithelial cell that undergoes an epithelial‐mesenchymal transition in forming the chondro‐osseous progenitor (COP) that expresses both osteoblast and chondrocyte transcripts. Red arrows show the fluorescence‐activated cell sorting isolation of either TR+ RSCs or TR+ COPs and the injection of 3000 of each TR+ cell into wild‐type mice undergoing heterotopic ossification.

The formation of neuromas involves expansion of the cellular components of peripheral nerves. The onset of these disorganized tumors involves activation of sensory nerves and neuroinflammation. Particularly problematic in neuroma is arborization of axons leading to extreme, neuropathic pain. The most common sites for neuroma are the ends of transected nerves following injury; however, this rodent model does not reliably result in neuroma formation. In this study, we established a rodent model of neuroma in which the sciatic nerve was loosely ligated with two chromic gut sutures. This model formed neuromas reliably (∼95%), presumably through activation of the neural inflammatory cascade. Resulting neuromas had a disorganized structure and a significant number of replicating cells. Quantification of changes in perineurial and Schwann cells showed a significant increase in these populations. Immunohistochemical analysis showed the presence of β-tubulin 3 in the rapidly expanding nerve and a decrease in neurofilament heavy chain compared to the normal nerve, suggesting the axons forming a disorganized structure. Measurement of the permeability of the blood–nerve barrier shows that it opened almost immediately and remained open as long as 10 days. Studies using an antagonist of the β3-adrenergic receptor (L-748,337) or cromolyn showed a significant reduction in tumor size and cell expansion as determined by flow cytometry, with an improvement in the animal’s gait detected using a Catwalk system. Previous studies in our laboratory have shown that heterotopic ossification is also a result of the activation of neuroinflammation. Since heterotopic ossification and neuroma often occur together in amputees, they were induced in the same limbs of the study animals. More heterotopic bone was formed in animals with neuromas as compared to those without. These data collectively suggest that perturbation of early neuroinflammation with compounds such as L-748,337 and cromolyn may reduce formation of neuromas.

Bone metastases occur in the majority of advanced breast cancer patients, and the most common complications are osteolytic bone metastases. However, due to the limitations of models and methodologies for bone metastasis studies, the intricacies of bone metastasis have not been fully acknowledged and revealed in prior work. Our earlier study indicated that certain breast cancer cells undergo a pre-osteolytic stage before the establishment of overt metastatic lesions. Here, we further identify a differential bone morphology between ER (estrogen receptor) + and ER − breast cancer. Specifically, we observe a more pronounced osteolytic phenotype in the bone metastatic lesions of ER − cells investigated, linked to elevated hypoxia signaling that stimulates the secretion of osteolytic inducers from cancer cells. In the in vivo mouse model, the application of the hypoxia-inducible factor (HIF) inhibitor 2-methoxyestradiol demonstrates a promising efficacy in suppressing tumor growth and osteoclast differentiation in the bone lesions established by bone-tropic subpopulation of ER − MDA-MB-231 cell. Overall, our findings explore the complexity of phenotype and morphology in bone metastatic lesions of ER + and ER − breast cancer, which offers a compelling rationale for leveraging HIF inhibitors to the treatment targeting skeletal complications of breast cancer bone metastases, especially for ER − tumors. A phenotypic study to examine the distinct morphologies of ER+ and ER− breast cancer bone metastases focusing on the mechanism associated with the hypoxia pathway and revealing a promising target for bone metastases treatment in breast cancer.

Osteoblasts are continually recruited from stem cell pools to maintain bone. Although their immediate precursor is a plastic-adherent 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.

Introduction: Motorcycles have become an increasingly popular mode of transportation despite their association with a greater risk for injury compared with automobiles. Whereas the recent incidence of annual passenger vehicle fatalities in the United States of America (USA) has progressively declined, motorcycle fatalities have steadily increased in the past 11 years. Although motorcycle injuries (MIs) have been studied, to the author's knowledge there are no published reports on MIs in the USA during this 11-year period. Methods : Study data were derived from a prospectively collected Level I trauma center database. Data sampling included motorcycle crash injury evaluations for the 10-year period ending on 31 August 2008. This retrospective analysis included patient demographic and medical data, helmet use, Glasgow coma scale (GCS) score, injury severity score (ISS), length of hospital stay (LOS), specific injury diagnosis, and death. Data statistics were analyzed using the Spearman correlation coefficient, Kruskal-Wallis tests, and logistic regression. Results: The study identified 1252 motorcycle crash injuries. Helmets were worn by 40.7% of patients for which helmet data were available. The rates of the most common orthopedic injuries were tibia/fibula (19.01%), spine (16.21%), and forearm (10.14%) fractures. The most common non-orthopedic motorcycle crash injuries were concussions (21.09%), skull fractures (8.23%), face fractures (13.66%), and hemo- and pneumothorax (8.79%). There was a significant correlation between greater age and higher ISS (r=0.21, P<0.0001) and longer LOS (r=0.22, P<0.0001). Older patients were also less likely to wear a helmet (OR=0.99, 95% CI: 0.98, 0.997), associated with a significantly higher risk for death (after adjustment for helmet use OR=1.03, 95% CI: 1.00, 1.05). All patients without helmets had a significantly lower GCS score (P=0.0001) and a higher mortality rate (after adjustment for patient demographic data OR=2.28, 95% CI: 1.13, 4.58).  Conclusion : Compared with historical reports, the prevalence of skull, face, spine, and pelvis fractures have increased in American motorcycle crashes. Compared to recent European studies, the incidence of USA skull and face fractures is much higher, while the incidence of USA spine and pelvis fractures is more comparable; however, this is not associated with increased in-hospital mortality.