Callus γδ T cells and microbe-induced intestinal Th17 cells improve fracture healing in mice.

IL-17A (IL-17), a driver of the inflammatory phase of fracture repair, is produced locally by several cell lineages including γδ T cells and Th17 cells. However, the origin of these T cells and their relevance for fracture repair are unknown. Here, we show that fractures rapidly expanded callus γδ T cells, which led to increased gut permeability by promoting systemic inflammation. When the microbiota contained the Th17 cell-inducing taxon segmented filamentous bacteria (SFB), activation of γδ T cells was followed by expansion of intestinal Th17 cells, their migration to the callus, and improved fracture repair. Mechanistically, fractures increased the S1P receptor 1-mediated (S1PR1-mediated) egress of Th17 cells from the intestine and enhanced their homing to the callus through a CCL20-mediated mechanism. Fracture repair was impaired by deletion of γδ T cells, depletion of the microbiome by antibiotics (Abx), blockade of Th17 cell egress from the gut, or Ab neutralization of Th17 cell influx into the callus. These findings demonstrate the relevance of the microbiome and T cell trafficking for fracture repair. Modifications of microbiome composition via Th17 cell-inducing bacteriotherapy and avoidance of broad-spectrum Abx may represent novel therapeutic strategies to optimize fracture healing.

Impact of Alcohol on Bone Health in People Living With HIV: Integrating Clinical Data From Serum Bone Markers With Morphometric Analysis in a Non-Human Primate Model.

People living with HIV (PLWH) represent a vulnerable population to adverse musculoskeletal outcomes due to HIV infection, antiretroviral therapy (ART), and at-risk alcohol use. Developing measures to prevent skeletal degeneration in this group requires a grasp of the relationship between alcohol use and low bone mass in both the PLWH population and its constituents as defined by sex, age, and race. We examined the association of alcohol use with serum biochemical markers of bone health in a diverse cohort of PLWH enrolled in the New Orleans Alcohol Use in HIV (NOAH) study. To explore the effects of alcohol on bone in the context of HIV and ART and the role of estrogen, we conducted a parallel, translational study using simian immunodeficiency virus (SIV)+/ART+ female rhesus macaques divided into four groups: vehicle (Veh)/Sham; chronic binge alcohol (CBA)/Sham; Veh/ovariectomy (OVX); and CBA/OVX. Clinical data showed that both osteocalcin (Ocn) and procollagen type I N-propeptide (PINP) levels were inversely associated with multiple measures of alcohol consumption. Age (>50 years) significantly increased susceptibility to alcohol-associated suppression of bone formation in both female and male PLWH, with postmenopausal status appearing as an additional risk factor in females. Serum sclerostin (Scl) levels correlated positively with measures of alcohol use and negatively with Ocn. Micro-CT analysis of the macaque tibias revealed that although both CBA and OVX independently decreased trabecular number and bone mineral density, only OVX decreased trabecular bone volume fraction and impacted cortical geometry. The clinical data implicate circulating Scl in the pathogenesis of alcohol-induced osteopenia and suggest that bone morphology can be significantly altered in the absence of net change in osteoblast function as measured by serum markers. Inclusion of sophisticated tools to evaluate skeletal strength in clinical populations will be essential to understand the impact of alcohol-induced changes in bone microarchitecture. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

The microbiome restrains melanoma bone growth by promoting intestinal NK and Th1 cell homing to bone.

Bone metastases are frequent complications of malignant melanoma leading to reduced quality of life and significant morbidity. Regulation of immune cells by the gut microbiome influences cancer progression, but the role of the microbiome in tumor growth in bone is unknown. Using intracardiac or intratibial injections of B16-F10 melanoma cells into mice, we showed that gut microbiome depletion by broad-spectrum antibiotics accelerated intraosseous tumor growth and osteolysis. Microbiome depletion blunted melanoma-induced expansion of intestinal NK cells and Th1 cells and their migration from the gut to tumor-bearing bones. Demonstrating the functional relevance of immune cell trafficking from the gut to the bone marrow (BM) in bone metastasis, blockade of S1P-mediated intestinal egress of NK and Th1 cells, or inhibition of their CXCR3/CXCL9-mediated influx into the BM, prevented the expansion of BM NK and Th1 cells and accelerated tumor growth and osteolysis. Using a mouse model, this study revealed mechanisms of microbiota-mediated gut-bone crosstalk that are relevant to the immunological restraint of melanoma metastasis and tumor growth in bone. Microbiome modifications induced by antibiotics might have negative clinical consequences in patients with melanoma.

Temporospatial regulation of intraflagellar transport is required for the endochondral ossification in mice.

Ciliogenic components, such as the family of intraflagellar transport (IFT) proteins, are recognized to play key roles in endochondral ossification, a critical process to form most bones. However, the unique functions and roles of each IFT during endochondral ossification remain unclear. Here, we show that IFT20 is required for endochondral ossification in mice. Utilizing osteo-chondrocyte lineage-specific Cre mice (Prx1-Cre and Col2-Cre), we deleted Ift20 to examine its function. Although chondrocyte-specific Ift20 deletion with Col2-Cre mice did not cause any overt skeletal defects, mesoderm-specific Ift20 deletion using Prx1-Cre (Ift20:Prx1-Cre) mice resulted in shortened limb outgrowth. Primary cilia were absent on chondrocytes of Ift20:Prx1-Cre mice, and ciliary-mediated Hedgehog signaling was attenuated in Ift20:Prx1-Cre mice. Interestingly, loss of Ift20 also increased Fgf18 expression in the perichondrium that sustained Sox9 expression, thus preventing endochondral ossification. Inhibition of enhanced phospho-ERK1/2 activation partially rescued defective chondrogenesis in Ift20 mutant cells, supporting an important role for FGF signaling. Our findings demonstrate that IFT20 is a critical regulator of temporospatial FGF signaling that is required for endochondral ossification.

MyD88 Is Not Required for Muscle Injury-Induced Endochondral Heterotopic Ossification in a Mouse Model of Fibrodysplasia Ossificans Progressiva.

Excess inflammation and canonical BMP receptor (BMPR) signaling are coinciding hallmarks of the early stages of injury-induced endochondral heterotopic ossification (EHO), especially in the rare genetic disease fibrodysplasia ossificans progressiva (FOP). Multiple inflammatory signaling pathways can synergistically enhance BMP-induced Smad1/5/8 activity in multiple cell types, suggesting the importance of pathway crosstalk in EHO and FOP. Toll-like receptors (TLRs) and IL-1 receptors mediate many of the earliest injury-induced inflammatory signals largely via MyD88-dependent pathways. Thus, the hypothesis that MyD88-dependent signaling is required for EHO was tested in vitro and in vivo using global or Pdgfrα-conditional deletion of MyD88 in FOP mice. As expected, IL-1ß or LPS synergistically increased Activin A (ActA)-induced phosphorylation of Smad 1/5 in fibroadipoprogenitors (FAPs) expressing Alk2R206H. However, conditional deletion of MyD88 in Pdgfrα-positive cells of FOP mice did not significantly alter the amount of muscle injury-induced EHO. Even more surprisingly, injury-induced EHO was not significantly affected by global deletion of MyD88. These studies demonstrate that MyD88-dependent signaling is dispensable for injury-induced EHO in FOP mice.

Do Interactions of Vitamin D3 and BMP Signaling Hold Implications in the Pathogenesis of Fibrodysplasia Ossificans Progressiva?

PURPOSE OF REVIEW: Fibrodysplasia ossificans progressiva (FOP) is a debilitating rare disease known for episodic endochondral heterotopic ossification (HO) caused by gain-of-function mutations in ACVR1/ALK2. However, disease severity varies among patients with identical mutations suggesting disease-modifying factors, including diet, may have therapeutic implications. The roles of vitamin D3 in calcium metabolism and chondrogenesis are known, but its effects on BMP signaling and chondrogenesis are less studied. This review attempts to assess the possibility of vitamin D's effects in FOP by exploring relevant intersections of VD3 with mechanisms of FOP flares. RECENT FINDINGS: In vitro and in vivo studies suggest vitamin D suppresses inflammation, while clinical studies suggest that vitamin D3 protects against arteriosclerosis and inversely correlates with non-genetic intramuscular HO. However, the enhancement of chondrogenesis, BMP signaling, and possibly Activin A expression by vitamin D may be more relevant in FOP. There appears to be little potential for vitamin D to reduce HO in FOP, but testing the potential for excess vitamin D to promote HO may be warranted.

The Role of Bone Morphogenetic Protein Signaling in Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) affects over 30% of adults in the United States. Bone morphogenetic protein (BMP) signaling is known to contribute to hepatic fibrosis, but the role of BMP signaling in the development of NAFLD is unclear. In this study, treatment with either of two BMP inhibitors reduced hepatic triglyceride content in diabetic (db/db) mice. BMP inhibitor-induced decrease in hepatic triglyceride levels was associated with decreased mRNA encoding Dgat2, an enzyme integral to triglyceride synthesis. Treatment of hepatoma cells with BMP2 induced DGAT2 expression and activity via intracellular SMAD signaling. In humans we identified a rare missense single nucleotide polymorphism in the BMP type 1 receptor ALK6 (rs34970181;R371Q) associated with a 2.1-fold increase in the prevalence of NAFLD. In vitro analyses revealed R371Q:ALK6 is a previously unknown constitutively active receptor. These data show that BMP signaling is an important determinant of NAFLD in a murine model and is associated with NAFLD in humans.

Drill and Fill Technique for the Treatment of Scaphoid Delayed Unions and Nonunions.

Background This article reviews the results of a surgical technique using three iterations of drilling , autologous cancellous bone grafting ( filling ), and use of an intraosseous compression screw for the treatment of nondisplaced or minimally displaced scaphoid delayed unions or nonunions. Methods Part 1-Cadaveric study: Three cadaveric scaphoids underwent stained cancellous bone graft packing and headless cannulated compression screw placement using a single iteration of drilling and graft packing. Three additional scaphoids were allocated to the triple "drill and fill" group, and underwent three iterations of drilling and graft packing before screw insertion. Graft particle distribution on mid-sagittal sections was assessed under fluorescence microscopy. Comparison of normalized areas between the single and triple "drill and fill" groups was performed using repeated measures ANOVA and Tukey's post hoc test. Part 2-Clinical study: Twelve patients with minimally displaced scaphoid delayed unions and nonunions treated between April 2007 and December 2013 with the triple "drill and fill" technique were included. The average follow-up was 60.4 weeks. Two fellowship-trained musculoskeletal radiologists independently reviewed images for fracture healing. Results By the histomorphometric analysis, there was improved autograft distribution along the screw tract, particularly within the proximal pole, with three iterations of drilling and filling. Clinically, 11 of 12 delayed unions and nonunions had healed. Conclusion Our results support the use of the "drill and fill" technique as an option for the treatment of select nondisplaced or minimally displaced scaphoid nonunions and delayed unions at the waist without avascular necrosis of the proximal pole. Level of Evidence This is a Level IV study.

Methods for the reliable induction of heterotopic ossification in the conditional Alk2Q207D mouse.

OBJECTIVES: Conditional Alk2Q207D-floxed (caALK2fl) mice have previously been used as a model of heterotopic ossification (HO). However, HO formation in this model can be highly variable, and it is unclear which methods reliably induce HO. Hence, these studies report validated methods for reproducibly inducing HO in caALK2fl mice. METHODS: Varying doses of Adex-cre and cardiotoxin (CTX) were injected into the calf muscles of 9, 14, or 28-day-old caALK2fl/- or caALK2fl/fl mice. HO was measured by planar radiography or microCT at 14-28 days post-injury. RESULTS: In 9-day-old caALK2fl/- or caALK2fl/fl mice, single injections of 109 PFU Adex-cre and 0.3 µg of CTX were sufficient to induce extensive HO within 14 days post-injury. In 28-day-old mice, the doses were increased to 5 x 109 PFU Adex-cre and 3.0 µg of CTX to achieve similar consistency, but at a slower rate versus younger mice. Using a crush injury, instead of CTX, also provided consistent induction of HO. Finally, the Type 1 BMPR inhibitor, DMH1, significantly reduced HO formation in 28-day-old caALK2fl/fl mice. CONCLUSIONS: These data illustrate multiple methods for reliable induction of localized HO in the caALK2flmouse that can serve as a starting point for new laboratories utilizing this model.

LNK deficiency promotes acute aortic dissection and rupture.

Aortic dissection (AD) is a life-threatening vascular disease with limited treatment strategies. Here, we show that loss of the GWAS-identified SH2B3 gene, encoding lymphocyte adaptor protein LNK, markedly increases susceptibility to acute AD and rupture in response to angiotensin (Ang) II infusion. As early as day 3 following Ang II infusion, prior to the development of AD, Lnk-/- aortas display altered mechanical properties, increased elastin breaks, collagen thinning, enhanced neutrophil accumulation, and increased MMP-9 activity compared with WT mice. Adoptive transfer of Lnk-/- leukocytes into Rag1-/- mice induces AD and rupture in response to Ang II, demonstrating that LNK deficiency in hematopoietic cells plays a key role in this disease. Interestingly, treatment with doxycycline prevents the early accumulation of aortic neutrophils and significantly reduces the incidence of AD and rupture. PrediXcan analysis in a biobank of more than 23,000 individuals reveals that decreased expression of SH2B3 is significantly associated with increased frequency of AD-related phenotypes (odds ratio 0.81). Thus, we identified a role for LNK in the pathology of AD in experimental animals and humans and describe a new model that can be used to inform both inherited and acquired forms of this disease.

The proto-oncogene function of Mdm2 in bone.

Mouse double minute 2 (Mdm2) is a multifaceted oncoprotein that is highly regulated with distinct domains capable of cellular transformation. Loss of Mdm2 is embryonically lethal, making it difficult to study in a mouse model without additional genetic alterations. Global overexpression through increased Mdm2 gene copy number (Mdm2Tg ) results in the development of hematopoietic neoplasms and sarcomas in adult animals. In these mice, we found an increase in osteoblastogenesis, differentiation, and a high bone mass phenotype. Since it was difficult to discern the cell lineage that generated this phenotype, we generated osteoblast-specific Mdm2 overexpressing (Mdm2TgOb ) mice in 2 different strains, C57BL/6 and DBA. These mice did not develop malignancies; however, these animals and the MG63 human osteosarcoma cell line with high levels of Mdm2 showed an increase in bone mineralization. Importantly, overexpression of Mdm2 corrected age-related bone loss in mice, providing a role for the proto-oncogenic activity of Mdm2 in bone health of adult animals.

Differences in sensitivity to microstructure between cyclic- and impact-based microindentation of human cortical bone.

Unlike the known relationships between traditional mechanical properties and microstructural features of bone, the factors that influence the mechanical resistance of bone to cyclic reference point microindention (cRPI) and impact microindention (IMI) have yet to be identified. To determine whether cRPI and IMI properties depend on microstructure, we indented the tibia mid-shaft, the distal radius, and the proximal humerus from 10 elderly donors using the BioDent and OsteoProbe (neighboring sites). As the only output measure of IMI, bone material strength index (BMSi) was significantly different across all three anatomical sites being highest for the tibia mid-shaft and lowest for the proximal humerus. Total indentation distance (inverse of BMSi) was higher for the proximal humerus than for the tibia mid-shaft but was not different between other anatomical comparisons. As a possible explanation for the differences in BMSi, pore water, as determined by 1 H nuclear magnetic resonance, was lowest for the tibia and highest for the humerus. Moreover, the local intra-cortical porosity, as determined by micro-computed tomography, was negatively correlated with BMSi for both arm bones. BMSi was also positively correlated with peak bending stress of cortical bone extracted from the tibia mid-shaft. Microstructural correlations with cRPI properties were not significant for any of the bones. The one exception was that average energy dissipated during cRPI was negatively correlated with local tissue mineral density in the tibia mid-shaft. With higher indentation force and larger tip diameter than cRPI, only IMI appears to be sensitive to the underlying porosity of cortical bone. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1442-1452, 2017.

Novel methods for microCT-based analyses of vasculature in the renal cortex reveal a loss of perfusable arterioles and glomeruli in eNOS-/- mice.

BACKGROUND: Two-dimensional measures of vascular architecture provide incomplete information about vascular structure. This study applied a novel rigorous method for 3D microCT-based analysis of total and cortical renal vasculature combined with a novel method to isolate and quantify the number of perfused glomeruli to assess vascular changes in eNOS-/- mice. METHODS: Two month old male wildtype and eNOS-/- mice were perfused with heparinized saline followed by radiopaque Microfil. The Microfil-perfused vasculature of excised kidneys was imaged by µCT with an isotropic voxel-size of 5.0 µm. For analysis of renal cortical vasculature, a custom algorithm was created to define the cortical volume of interest (VOI) as the entire volume within 600 µm of the renal surface. Vessel thickness in the whole kidney or renal cortex was analyzed by plotting the distribution of vascular volume at each measured thickness and examining differences between the genotypes at individual thicknesses. A second image processing algorithm was created to isolate, identify, and extract contrast perfused glomeruli from the cortical vessels. RESULTS: Fractional vascular volume (vascular volume/kidney volume; VV/KV) and Vessel Number/mm (V.N) were significantly lower in eNOS-/- mice vs. WT (p < 0.05). eNOS-/- kidneys had significantly fewer perfusable vessels vs. WT in the range of 20-40 µm in thickness. The cortex of eNOS-/- kidneys had significantly lower VV, VV/cortical volume, and V.N, with an increase in the distance between vessels (all p < 0.05). The total volume of vessels in the range of 20-30 µm was significantly lower in the cortex of eNOS-/- mice compared to WT (p < 0.05). Moreover, the total number of perfused glomeruli was significantly decreased in eNOS-/- mice (p < 0.01). CONCLUSIONS: The methods presented here demonstrate a new method to analyze contrast enhanced µCT images for vascular phenotyping of the murine kidney. These data also demonstrate that kidneys in eNOS-/- mice have severe defects in vascular perfusion/structure in the renal cortex.

Predicting mouse vertebra strength with micro-computed tomography-derived finite element analysis.

As in clinical studies, finite element analysis (FEA) developed from computed tomography (CT) images of bones are useful in pre-clinical rodent studies assessing treatment effects on vertebral body (VB) strength. Since strength predictions from microCT-derived FEAs (µFEA) have not been validated against experimental measurements of mouse VB strength, a parametric analysis exploring material and failure definitions was performed to determine whether elastic µFEAs with linear failure criteria could reasonably assess VB strength in two studies, treatment and genetic, with differences in bone volume fraction between the control and the experimental groups. VBs were scanned with a 12-µm voxel size, and voxels were directly converted to 8-node, hexahedral elements. The coefficient of determination or R (2) between predicted VB strength and experimental VB strength, as determined from compression tests, was 62.3% for the treatment study and 85.3% for the genetic study when using a homogenous tissue modulus (E t) of 18 GPa for all elements, a failure volume of 2%, and an equivalent failure strain of 0.007. The difference between prediction and measurement (that is, error) increased when lowering the failure volume to 0.1% or increasing it to 4%. Using inhomogeneous tissue density-specific moduli improved the R (2) between predicted and experimental strength when compared with uniform E t=18 GPa. Also, the optimum failure volume is higher for the inhomogeneous than for the homogeneous material definition. Regardless of model assumptions, µFEA can assess differences in murine VB strength between experimental groups when the expected difference in strength is at least 20%.

Combined MEK inhibition and BMP2 treatment promotes osteoblast differentiation and bone healing in Nf1Osx -/- mice.

Neurofibromatosis type I (NF1) is an autosomal dominant disease with an incidence of 1/3000, caused by mutations in the NF1 gene, which encodes the RAS/GTPase-activating protein neurofibromin. Non-bone union after fracture (pseudarthrosis) in children with NF1 remains a challenging orthopedic condition to treat. Recent progress in understanding the biology of neurofibromin suggested that NF1 pseudarthrosis stems primarily from defects in the bone mesenchymal lineage and hypersensitivity of hematopoietic cells to TGFß. However, clinically relevant pharmacological approaches to augment bone union in these patients remain limited. In this study, we report the generation of a novel conditional mutant mouse line used to model NF1 pseudoarthrosis, in which Nf1 can be ablated in an inducible fashion in osteoprogenitors of postnatal mice, thus circumventing the dwarfism associated with previous mouse models where Nf1 is ablated in embryonic mesenchymal cell lineages. An ex vivo-based cell culture approach based on the use of Nf1(flox/flox) bone marrow stromal cells showed that loss of Nf1 impairs osteoprogenitor cell differentiation in a cell-autonomous manner, independent of developmental growth plate-derived or paracrine/hormonal influences. In addition, in vitro gene expression and differentiation assays indicated that chronic ERK activation in Nf1-deficient osteoprogenitors blunts the pro-osteogenic property of BMP2, based on the observation that only combination treatment with BMP2 and MEK inhibition promoted the differentiation of Nf1-deficient osteoprogenitors. The in vivo preclinical relevance of these findings was confirmed by the improved bone healing and callus strength observed in Nf1osx (-/-) mice receiving Trametinib (a MEK inhibitor) and BMP2 released locally at the fracture site via a novel nanoparticle and polyglycidol-based delivery method. Collectively, these results provide novel evidence for a cell-autonomous role of neurofibromin in osteoprogenitor cells and insights about a novel targeted approach for the treatment of NF1 pseudoarthrosis.

Inner Ear Vestibular Signals Regulate Bone Remodeling via the Sympathetic Nervous System.

The inner ear vestibular system has numerous projections on central brain centers that regulate sympathetic outflow, and skeletal sympathetic projections affect bone remodeling by inhibiting bone formation by osteoblasts and promoting bone resorption by osteoclasts. In this study, we show that bilateral vestibular lesions in mice cause a low bone mass phenotype associated with decreased bone formation and increased bone resorption. This reduction in bone mass is most pronounced in lower limbs, is not associated with reduced locomotor activity or chronic inflammation, and could be prevented by the administration of the ß-blocker propranolol and by genetic deletion of the ß2-adrenergic receptor, globally or specifically in osteoblasts. These results provide novel experimental evidence supporting a functional autonomic link between central proprioceptive vestibular structures and the skeleton. Because vestibular dysfunction often affects the elderly, these results also suggest that age-related bone loss might have a vestibular component and that patients with inner ear pathologies might be at risk for fracture. Lastly, these data might have relevance to the bone loss observed in microgravity, as vestibular function is altered in this condition as well. © 2015 American Society for Bone and Mineral Research.

Asfotase-α improves bone growth, mineralization and strength in mouse models of neurofibromatosis type-1.

Individuals with neurofibromatosis type-1 (NF1) can manifest focal skeletal dysplasias that remain extremely difficult to treat. NF1 is caused by mutations in the NF1 gene, which encodes the RAS GTPase-activating protein neurofibromin. We report here that ablation of Nf1 in bone-forming cells leads to supraphysiologic accumulation of pyrophosphate (PPi), a strong inhibitor of hydroxyapatite formation, and that a chronic extracellular signal-regulated kinase (ERK)-dependent increase in expression of genes promoting PPi synthesis and extracellular transport, namely Enpp1 and Ank, causes this phenotype. Nf1 ablation also prevents bone morphogenic protein-2-induced osteoprogenitor differentiation and, consequently, expression of alkaline phosphatase and PPi breakdown, further contributing to PPi accumulation. The short stature and impaired bone mineralization and strength in mice lacking Nf1 in osteochondroprogenitors or osteoblasts can be corrected by asfotase-α enzyme therapy aimed at reducing PPi concentration. These results establish neurofibromin as an essential regulator of bone mineralization. They also suggest that altered PPi homeostasis contributes to the skeletal dysplasias associated with NF1 and that some of the NF1 skeletal conditions could be prevented pharmacologically.

Development of Raman spectral markers to assess metastatic bone in breast cancer.

Bone is the most common site for breast cancer metastases. One of the major complications of bone metastasis is pathological bone fracture caused by chronic bone loss and degeneration. Current guidelines for the prediction of pathological fracture mainly rely on radiographs or computed tomography, which are limited in their ability to predict fracture risk. The present study explored the feasibility of using Raman spectroscopy to estimate pathological fracture risk by characterizing the alterations in the compositional properties of metastatic bones. Tibiae with evident bone destruction were investigated using Raman spectroscopy. The carbonation level calculated by the ratio of carbonate/phosphate ν1 significantly increased in the tumor-bearing bone at all the sampling regions at the proximal metaphysis and diaphysis, while tumor-induced elevation in mineralization and crystallinity was more pronounced in the metaphysis. Furthermore, the increased carbonation level is positively correlated to bone lesion size, indicating that this parameter could serve as a unique spectral marker for tumor progression and bone loss. With the promising advances in the development of spatially offset Raman spectroscopy for deep tissue measurement, this spectral marker can potentially be used for future noninvasive evaluation of metastatic bone and prediction of pathological fracture risk.

Prostate cancer metastases alter bone mineral and matrix composition independent of effects on bone architecture in mice--a quantitative study using microCT and Raman spectroscopy.

Prostate cancer is the most common primary tumor and the second leading cause of cancer-related deaths in men in the United States. Prostate cancer bone metastases are characterized by abnormal bone remodeling processes and result in a variety of skeletal morbidities. Prevention of skeletal complications is a crucial element in prostate cancer management. This study investigated prostate cancer-induced alterations in the molecular composition and morphological structure of metastasis-bearing bones in a mouse model of prostate cancer using Raman spectroscopy and micro-computed tomography (microCT). LNCaP C4-2B prostate cancer cells were injected into the right tibiae of 5-week old male SCID mice. Upon sacrifice at 8weeks post tumor inoculation, two out of the ten tumor-bearing tibiae showed only osteoblastic lesions in the radiographs, 4 osteolytic lesions only and 4 mixed with osteoblastic and osteolytic lesions. Carbonate substitution was significantly increased while there was a marked reduction in the level of collagen mineralization, mineral crystallinity, and carbonate:matrix ratio in the cortex of the intact tumor-bearing tibiae compared to contralateral controls. MicroCT analysis revealed a significant reduction in bone volume/total volume, trabecular number and trabecular thickness, as well as significant increase in bone surface/volume ratio in tibiae with osteolytic lesions, suggesting active bone remodeling and bone loss. None of the changes in bone compositional properties were correlated with lesion area from radiographs or the changes in bone architecture from microCT. This study indicates that LNCaP C4-2B prostate cancer metastases alter bone tissue composition independent of changes in architecture, and altered bone quality may be an important contributor to fracture risk in these patients. Raman spectroscopy may provide a new avenue of investigation into interactions between tumor and bone microenvironment.