Quantitative evaluation of correlation of dose and FDG-PET uptake value with clinical chest wall complications in patients with lung cancer treated with stereotactic body radiation therapy.

The aim of this study was to investigate quantitatively the dosimetric factors that increase the risk of clinical complications of rib fractures or chest wall pain after stereotactic body radiation therapy (SBRT) to the lung. The correlations of clinical complications with standard-uptake values (SUV) and FDG-PET activity distributions from post-treatment PET-imaging were studied. Mean and maximum doses from treatment plans, FDG-PET activity values on post-SBRT PET scans and the presence of clinical complications were determined in fifteen patients undergoing 16 SBRT treatments for lung cancer. SBRT treatments were delivered in 3 to 5 fractions using 5 to 7 fields to prescription doses in the range from 39.0 to 60.0 Gy. The dose and FDG-PET activity values were extracted from regions of interest in the chest wall that matched anatomically. Quantitative evaluation of the correlation between dose deposition and FDG-PET activity was performed by calculating the Pearson correlation coefficient using pixel-by-pixel analysis of dose and FDG-PET activity maps in selected regions of interest associated with clinical complications. Overall, three of fifteen patients developed rib fractures with chest wall pain, and two patients developed pain symptoms without fracture. The mean dose to the rib cage in patients with fractures was 37.53 Gy compared to 33.35 Gy in patients without fractures. Increased chest wall activity as determined by FDG-uptake was noted in patients who developed rib fractures. Enhanced activity from PET-images correlated strongly with high doses deposited to the chest wall which could be predicted by a linear relationship. The local enhanced activity was associated with the development of clinical complications such as chest wall inflammation and rib fracture. This study demonstrates that rib fractures and chest wall pain can occur after SBRT treatments to the lung and is associated with increased activity on subsequent PET scans. The FDG-PET activity provides a useful parameter that can be used clinically to predict chest wall complication in lung patients.

Sonic hedgehog regulates osteoblast function by focal adhesion kinase signaling in the process of fracture healing.

Several biological studies have indicated that hedgehog signaling plays an important role in osteoblast proliferation and differentiation, and sonic hedgehog (SHH) expression is positively correlated with phosphorylated focal adhesion kinase (FAK) Tyr(397). However, the relationship between them and their role in the process of normal fracture repair has not been clarified yet. Immunohistochemical analysis revealed that SHH and pFAK Tyr(397) were expressed in bone marrow cells and that pFAK Tyr(397) was also detected in ALP-positive osteoblasts near the TRAP-positive osteoclasts in the fracture site in the ribs of mice on day 5 after fracture. SHH and pFAK Tyr(397) were detectable in osteoblasts near the hypertrophic chondrocytes on day 14. In vitro analysis showed that SHH up-regulated the expression of FAK mRNA and pFAK Tyr(397) time dependently in osteoblastic MC3T3-E1 cells. Functional analysis revealed that 5 lentivirus encoding short hairpin FAK RNAs (shFAK)-infected MC3T3-E1 cell groups displayed a round morphology and decreased proliferation, adhesion, migration, and differentiation. SHH stimulated the proliferation and differentiation of MC3T3-E1 cells, but had no effect on the shFAK-infected cells. SHH also stimulated osteoclast formation in a co-culture system containing MC3T3-E1 and murine CD11b(+) bone marrow cells, but did not affect the shFAK-infected MC3T3-E1 co-culture group. These data suggest that SHH signaling was activated in osteoblasts at the dynamic remodeling site of a bone fracture and regulated their proliferation and differentiation, as well as osteoclast formation, via FAK signaling.

Rib fractures and death from deletion of osteoblast ßcatenin in adult mice is rescued by corticosteroids.

Ribs are primarily made of cortical bone and are necessary for chest expansion and ventilation. Rib fractures represent the most common type of non-traumatic fractures in the elderly yet few studies have focused on the biology of rib fragility. Here, we show that deletion of ßcatenin in Col1a2 expressing osteoblasts of adult mice leads to aggressive osteoclastogenesis with increased serum levels of the osteoclastogenic cytokine RANKL, extensive rib resorption, multiple spontaneous rib fractures and chest wall deformities. Within days of osteoblast specific ßcatenin deletion, animals die from respiratory failure with a vanishing rib cage that is unable to sustain ventilation. Increased bone resorption is also observed in the vertebrae and femur. Treatment with the bisphosphonate pamidronate delayed but did not prevent death or associated rib fractures. In contrast, administration of the glucocorticoid dexamethasone decreased serum RANKL and slowed osteoclastogenesis. Dexamethasone preserved rib structure, prevented respiratory compromise and strikingly increased survival. Our findings provide a novel model of accelerated osteoclastogenesis, where deletion of osteoblast ßcatenin in adults leads to rapid development of destructive rib fractures. We demonstrate the role of ßcatenin dependent mechanisms in rib fractures and suggest that glucocorticoids, by suppressing RANKL, may have a role in treating bone loss due to aggressive osteoclastogenesis.

Anesthetic management of a horse with traumatic pneumothorax.

A traumatic pneumothorax and severe hemorrhage were present in a mare with a large thoracic wall defect, lung perforation, and multiple rib fractures. General anesthesia was induced to allow surgical exploration. We describe the anesthetic technique, and discuss the management of the ventilatory, hemodynamic, and metabolic disturbances encountered.

Stromal cell-derived factor 1 regulates the actin organization of chondrocytes and chondrocyte hypertrophy.

Stromal cell-derived factor 1 (SDF-1/CXCL12/PBSF) plays important roles in the biological and physiological functions of haematopoietic and mesenchymal stem cells. This chemokine regulates the formation of multiple organ systems during embryogenesis. However, its roles in skeletal development remain unclear. Here we investigated the roles of SDF-1 in chondrocyte differentiation. We demonstrated that SDF-1 protein was expressed at pre-hypertrophic and hypertrophic chondrocytes in the newly formed endochondral callus of rib fracture as well as in the growth plate of normal mouse tibia by immunohistochemical analysis. Using SDF-1(-/-) mouse embryo, we histologically showed that the total length of the whole humeri of SDF-1(-/-) mice was significantly shorter than that of wild-type mice, which was contributed mainly by shorter hypertrophic and calcified zones in SDF-1(-/-) mice. Actin cytoskeleton of hypertrophic chondrocytes in SDF-1(-/-) mouse humeri showed less F-actin and rounder shape than that of wild-type mice. Primary chondrocytes from SDF-1(-/-) mice showed the enhanced formation of philopodia and loss of F-actin. The administration of SDF-1 to primary chondrocytes of wild-type mice and SDF-1(-/-) mice promoted the formation of actin stress fibers. Organ culture of embryonic metatarsals from SDF-1(-/-) mice showed the growth delay, which was recovered by an exogenous administration of SDF-1. mRNA expression of type X collagen in metatarsals and in primary chondrocytes of SDF-1(-/-) mouse embryo was down-regulated while the administration of SDF-1 to metatarsals recovered. These data suggests that SDF-1 regulates the actin organization and stimulates bone growth by mediating chondrocyte hypertrophy.

Transient expression of myofibroblast-like cells in rat rib fracture callus.

BACKGROUND AND PURPOSE: We have previously shown that early fracture callus of rat rib has viscoelastic and contractile properties resembling those of smooth muscle. The cells responsible for this contractility have been hypothesized to be myofibroblast-like in nature. In soft-tissue healing, force generated by contraction of myofibroblasts promotes healing. Accordingly, we tried to identify myofibroblast-like cells in early fibrous callus. ANIMALS AND METHODS: Calluses from rat rib fractures were removed 7, 14, and 21 days after fracture and unfractured ribs acted as controls. All tissues were analyzed using qPCR and immunohistochemistry. We analyzed expression of smooth muscle- and myofibroblast-associated genes and proteins including alpha smooth muscle actin (αSMA), non-muscle myosin, fibronectin extra domain A variant (EDA-fibronectin), OB-cadherin, connexin-43, basic calponin (h1CaP), and h-caldesmon. RESULTS: In calluses at 7 days post-fracture, there were statistically significant increases in expression of αSMA mRNA (2.5 fold), h1CaP mRNA (2.1 fold), EDA-fibronectin mRNA (14 fold), and connexin-43 mRNA (1.8 fold) compared to unfractured ribs, and by 21 days post-fracture mRNA expression in calluses had decreased to levels approaching those in unfractured rib. Immunohistochemistry of 7 day fibrous callus localized calponin, EDA-fibronectin and co-immunolabeling of OB-cadherin and αSMA (thus confirming a myofibroblastic phenotype) within various cell populations. INTERPRETATION: This study provides further evidence that early rat rib callus is not only smooth muscle-like in nature but also contains a notable population of cells that have a distinct myofibroblastic phenotype. The presence of these cells indicates that in vivo contraction of early callus is a mechanism that may occur in fractures so as to facilitate healing, as it does in soft tissue wound repair.

Production of VEGF receptor 1 and 2 mRNA and protein during endochondral bone repair is differential and healing phase specific.

Physiological disturbances, including temporary hypoxia, are expected to drive angiogenesis during bone repair. Evidence suggests that the angiogenic ligand vascular endothelial growth factor (VEGF)-A plays an important role in this process. We characterized the expression of two receptors that are essential for mediating VEGF signaling, VEGFR1/Flt-1 and VEGFR2/Flk-1/KDR, in a mouse rib fracture model. Their mRNA and protein levels were assessed in four healing phases, which were characterized histologically as hemorrhage formation on postfracture day (PFD) 1, inflammatory response on PFD 3, initiation of callus development on PFD 7, and the presence of a mature callus on PFD 14. Transcript was detected for VEGFR1 and VEGFR2, as well as VEGF. While mRNA expression of VEGFR1 was monophasic throughout all healing phases, VEGFR2 showed a biphasic profile with significantly increased mRNA expression during callus formation and maturation. Expression of VEGF mRNA was characterized by a more gradual increase during callus formation. The protein level for VEGFR1 was below detection sensitivity during the initial healing phase. It was then restored to a stable level, detectable through the subsequent healing phases. Hence, the VEGFR1 protein levels partially mirrored the transcript expression profile. In comparison, the protein level of VEGFR2 increased gradually during the healing phases and peaked at callus maturation. This correlated well with the transcriptional expression of VEGFR2. Intact bone from age-matched male mice had considerable protein levels of VEGFR1 and VEGF, but no detectable VEGFR2. Together, these findings uncovered expression signatures of the VEGF-VEGFR axis in endochondral bone repair.

Abdominal muscle uptake on FDG PET secondary to multiple rib fractures and use of accessory muscles of respiration.

A 69-year-old man with a history of lung cancer was referred for FDG whole body positron emission tomography (PET) with computed tomography (CT) fusion. Three days before the study, the patient was involved in a motor vehicle accident (MVA). In addition to hypermetabolic lesions representing metastatic lung cancer, PET scan showed marked anterior and lateral abdominal muscle uptake. Multiple focal abnormalities were seen on the PET scan that corresponded to multiple rib fractures on the CT scan that were secondary to the MVA. When the patient was interviewed, he stated that he had thoracic wall pain that prevented him from breathing properly. This case illustrates an unusual pattern of increased anterior and lateral abdominal muscle uptake as a result of pain while breathing.

Expression of galanin and galanin receptor-1 in normal bone and during fracture repair in the rat.

The neuropeptide galanin (GAL) has recognized physiological actions in the nervous system and other tissues, but there is no documented evidence of GAL influencing normal or pathological bone metabolism. GAL expression, however, is upregulated in central and peripheral nerves following axotomy and is known to influence neural regeneration. Thus, severance of skeletal-associated nerves during fracture could similarly increase local GAL concentrations and thereby influence fracture healing. The initial aim of this study was therefore to identify the presence of GAL in normal bone and/or fracture callus by assessing the concentration and cellular localization of GAL in intact and/or fractured rat rib, using radioimmunoassay and immunohistochemistry, respectively. Groups of Sprague-Dawley rats (13 weeks old) had their left sixth ribs surgically fractured or underwent sham surgery and then calluses and nonfractured rib samples were analyzed at 1 and 2 weeks postsurgery (n = 5-6 per group). Low (basal) concentrations of GAL were detected in control ribs, whereas at 1 and 2 weeks postfracture, callus samples contained markedly increased levels of peptide ( approximately 32- and 18-fold increase, respectively, relative to controls; P < 0.01), revealing a strong upregulation during bone healing. Plasma GAL concentrations were also increased at 2 weeks postfracture (P < 0.005). In normal (nonfractured) rib, minimal levels of GAL-like immunoreactivity (LI) were present in cortical bone, periosteum, endosteum, and surrounding skeletal muscle. In costal cartilage plates, intense GAL-LI was present in all chondrocytes of the hypertrophic zone and in a population of chondrocytes in the reserve zone. GAL-LI was not present, however, in chondrocytes in the proliferative zone of costal cartilage or skeletal muscle fibers. In fracture callus, levels of GAL-LI were moderate to intense in osteoprogenitor cells and osteoblasts, in some chondrocytes, and in cartilaginous, osseous, and periosteal matrices. Subsequent studies revealed the presence of galanin receptor-1-like immunoreactivity (GALR1-LI) in most cell types shown to contain GAL-LI, although the distribution of GALR1-LI was more extensive in reserve zone chondrocytes than that of GAL-LI; and GALR1-LI also appeared in late proliferative zone chondrocytes of costal cartilage. In summary, GAL concentrations were significantly increased in fracture callus and plasma of rats that underwent rib fracture. In addition, GAL- and GALR1-LI was also detected in specific cells and structures within costal cartilage, bone, and fracture callus. These results strongly implicate GAL in aspects of cartilage growth plate physiology and fracture repair, possibly acting in an autocrine/paracrine fashion via GALR1.

F-18 FDG positron emission tomography and benign fractures.

PURPOSE: F-18 2-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET) has been used extensively in the imaging of cancer, including metastatic skeletal disease. Although uptake into benign osseous disease has been reported, there is very limited information regarding uptake into benign fractures. This report provides additional information regarding the appearance of benign fractures on FDG-PET images. MATERIALS AND METHODS: Four case reports of FDG-PET scanning are presented in patients with proved benign fractures. RESULTS: In three of these cases, FDG uptake was noted in fractures when images were obtained 17 days to 8 weeks after injury, with the most avid uptake observed when FDG-PET imaging was performed 17 days after fracture. In the patient in whom imaging was performed 8 weeks after fracture, no uptake of FDG was seen in a benign fracture. CONCLUSIONS: Fractures may accumulate FDG to varying degrees, and false-positive findings may occur when FDG-PET imaging is performed to assess for metastases, although the different pattern of uptake and clinical correlation usually allows accurate differentiation of fracture from skeletal metastases.

Bisphosphonate treatment suppresses not only stochastic remodeling but also the targeted repair of microdamage.

Two kinds of remodeling, stochastic and targeted, have been proposed based on the observation that microdamage in bone can initiate the remodeling process. Bisphosphonates are known to suppress stochastic bone remodeling. It has been hypothesized that bisphosphonates allow microdamage to accumulate, suggesting that they also suppress targeted remodeling. This study investigated whether suppression of remodeling using bisphosphonates inhibits remodeling targeted to repair microdamage, or whether the suppression of stochastic remodeling alone can account for the observed increase in damage accumulation. Beagle dogs were divided into three groups: control (CNT), risedronate-treated (RIS), and alendronate (ALN)-treated groups. The doses of both bisphosphonates were 6 times higher than the clinical doses. After 1-year treatment, animals were sacrificed and the right 9th rib was assigned to microdamage analysis. There were 3.06 times more associations between cracks and resorption spaces in CNT than expected (P < 0.005), indicating that remodeling normally targets cracks for repair, i.e., cracks can initiate a new remodeling event. However, although there was increased microdamage accumulation in RIS and ALN compared with CNT, fewer cracks than expected were associated with resorption spaces. The observation in RIS and ALN that there were fewer associations between cracks and resorption spaces than expected indicates that both targeted and non-targeted remodeling are suppressed in these groups. These data further suggest that the complete suppression of targeted remodeling could account for the increased microdamage burden.

Thoracic paravertebral block: radiological evidence of contralateral spread anterior to the vertebral bodies.

We report contralateral spread of contrast medium anterior to the vertebral bodies after injection of contrast through a thoracic paravertebral catheter that was used to manage pain in a patient with multiple fractured ribs. We review the literature and propose that the anatomical basis for this observation is spread in the extrapleural compartment of the thoracic paravertebral space along the subserous fascial plane.

Switch of osteonectin and osteopontin mRNA expression in the process of cartilage-to-bone transition during fracture repair.

The process of cartilage-to-bone transition (CBT) is a key event for the achievement of rigid bone healing during fracture repair. Since mineralization of cartilaginous matrix is a prerequisite for the initiation of CBT, the genetic localization of mineralization-related bone matrix proteins in CBT was examined in this study. An in situ hybridization method used on decalcified sections with digoxigenin-11-UTP labelled probes identified the cellular localizations of these genes in CBT. Cessation of osteonectin mRNA together with induction of osteopontin mRNA in chondrocyte maturation was observed during the process of CBT in the fracture callus on day 12 after fracture; osteocalcin mRNA was absent in chondrocytes of the CBT area. Induction of osteopontin mRNA in maturated chondrocytes was followed by the expression of mRNAs for osteonectin, osteopontin and osteocalcin in osteogenic cells in the ossification front of CBT. The data suggest that the switch from osteonectin to osteopontin mRNA expression in chondrocyte maturation is one of the key events during CBT. Transcriptional disorders of the expression of these molecules may be linked to the failure of fracture repair, i.e. delayed or prevented hypertrophic osteosynthesis.

[The experimental research of transforming growth factor-beta 1 expression during fracture healing].

OBJECTIVE: To observe the effect of TGF-beta 1 in the regulation of fracture healing. METHOD: The expression of transforming growth factor-beta 1 (TGF-beta 1) in different period of fracture healing was investigated by immunohistochemistry. RESULTS: It was found that the expression of TGF-beta 1 changed in different period. The cells in the cambial layer of the periostlum showed low or negative signal in the immediate injury response period. The osteoblasts differentiated from the periosteum cells stained strongly in the intramembranous ossification period, and the differentiated chondrocytes stained most strongly in the chondrogenesis period. The hypertrophic chondrocytes showed negative signal and the osteoblasts stained strongly in the endochondral ossification period. These results suggested that the expression of TGF-beta 1 was closely related to the proliferation and differentiation state of repair cells. CONCLUSION: TGF-beta 1 is intimately involved in the control of fracture healing.

Topical application of nerve growth factor improves fracture healing in rats.

The aim of the present study was to examine the effects of nerve growth factor on the healing of unsplinted fractured ribs. After fracture of a rib in male rats, nerve growth factor was delivered by a miniosmotic pump to the fracture site for 7 days at the rate of 1.4 micrograms/day. Callus catecholamine concentrations, bone callus size, histomorphometry, and biomechanical properties of the repairing rib were measured at 7, 21, and 42 days after fracture. After 21 days, concentrations of norepinephrine and epinephrine were significantly increased in the group treated with nerve growth factor compared with those in the control group (211% norepinephrine and 322% epinephrine). Also, the midline longitudinal area of non-osseous (fibrous tissue and cartilage) callus of the fracture was significantly smaller (54%) and had a higher proportion of cartilage in the treated group than in the controls. By 42 days, there was only bony callus between the fracture ends in both the control group and the treated group. The treated group, however, again showed significantly elevated concentrations of norepinephrine and epinephrine (286 and 382%, respectively) and significantly elevated breaking stress (50%) and Young's modulus (51%), together with a reduction in the transverse cross-sectional area of the repair site (57%). The resultant increases in effectiveness and rate of repair of bone with administration of nerve growth factor suggest that it may play an important role in the healing processes of fractured bone.

Transient and localized expression of bone morphogenetic protein 4 messenger RNA during fracture healing.

Temporal and spatial distribution of a gene encoding murine bone morphogenetic protein 4 (mBMP-4) during fracture repair were investigated in mice by RT-PCR and in situ hybridization. For in situ hybridization, fractured ribs and surrounding tissues were decalcified and hybridized with a mBMP-4-specific complementary RNA probe labeled with digoxigenin-11 UTP. mBMP-4 messenger RNA (mRNA) was not detected in ribs without fracture, whereas it was detected only in the early phase of fracture from 12 to 72 h after the onset of fracture before new cartilage or bone formation. The mBMP-4 mRNAs were present in cells distributed in three distinct regions, namely, the proliferating periosteum, the medullary cavity, and the muscles near the fracture site. These BMP-4-positive cells did not express bone gla protein mRNA, which is a marker of the mature osteogenic cell. RT-PCR also showed a transient increase in the level of BMP-4 mRNA in the early phase of fracture repair. The findings provide us with some new information. (1) The BMP-4 gene is produced by less differentiated osteoprogenitor cells, not by differentiated osteoblasts. (2) The BMP-4 gene is enhanced by the impact of fracture and localized in callus-forming tissue before callus formation. Together with the activities of BMP-4, as was previously described, our results suggest that newly produced BMP-4 gene product is one of the local contributing factors in callus formation in the early phase of fracture healing.

PGE2 induces regional remodeling changes in haversian envelope: a histomorphometric study of fractured ribs in beagles.

A histomorphometric study was carried out in the region of healing defects in the ribs of Beagles. A transverse fracture was made in the left 9th and 10th ribs. Eleven Beagles received either ethanol vehicle (n = 6) or PGE2 orally (n = 5) for the 30 day period from surgery to time of necropsy. Double-pulsed labels were given with each of two fluochrome markers, calcein prior to surgical treatment and oxytetracycline hydrochloride prior to euthanasia. The objectives were to determine the effects of fracture on regional remodeling in the haversian envelope; to determine the effects of PGE2 on the regional remodeling changes in the haversian envelope; and to determine the systemic effects of PGE2 on remodeling changes of the contralateral matching sites. The remodeling changes after surgically induced fracture were relatively insignificant between the injured and uninjured sides in the non-treated dogs as well as in the PGE2 treated group. This may be attributable to the global effect of a fracture. However, there were significantly slower rates of mineral apposition (P less than 0.05) and a longer period for osteon formation time (P less than 0.05) on the injured side compared to the uninjured side of the PGE2 treated dogs. The remodeling activities were increased on both sides in the PGE2 treated dogs when compared to the non-treated ones with more remodeling sites per unit cortical area (P less than 0.01), and a higher turnover rate (shorter total remodeling period in days) with a faster resorption rate and a net increase in mineral apposition rates at the end of the experiment (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Histochemical localization of calcium in the fracture callus with potassium pyroantimonate. Possible role of chondrocyte mitochondrial calcium in callus calcification.

Potassium pyroantimonate was employed as a histochemical stain for calcium at the ultrastructural level in the cartilaginous fracture callus in the rat rib. In areas of the callus showing no matrix mineralization, the electron-dense precipitate of the antimony-calcium complex was heavily deposited in chondrocyte mitochondria, lipid, and cell membrane. In areas showing early mineralization the mitochondria, lipid, and cell membrane showed a smaller amount of antimony-calcium complex, and in areas of more advanced matrix mineralization the mitochondria, lipid, and cell membrane were completely void of any stain. In the matrix, the initial site of mineralization was associated with matrix vesicles located in areas of early matrix mineralization. These findings suggest the hypothesis that mitochondria play an important role in matrix calcification in cartilaginous fracture callus.

Decreased 99mTc sulfur colloid activity in healed rib fractures.

This report describes 2 patients in whom focal areas of decreased 99mTc sulfur colloid marrow activity were associated with callus formation and healing rib fractures in one case and rib fractures with bony bridging in the second. Since bone marrow scans are occasionally used to select appropriate sites for marrow biopsy in patients with suspected metastatic disease, radiographic correlation of "cold" lesions on marrow scans is recommended prior to biopsy to exclude fracture with callus formation as a benign cause of the abnormality.

Tissue gas tensions and oxygen consumption in healing bone defects.

A technique for measuring the gaseous environment during the reconstitution of a large bone defect is described. Extensive testing of this system over the past 10 years has demonstrated its validity in measuring the average extracellular tissue pO-2 and pCO-2. Histology of the tissue surrounding the tonometer was obtained and correlated with the gaseous measurements. New bone formation in the healing segmental bone defects has been shown to take place under hypoxic conditions. The oxygen consumption of the surrounding tissue was determined and correlated with the histology and tissue gas measurements. It was demonstrated that the oxygen consumption was not elevated during the period of new bone formation, reflecting a state of anaerobic metabolism. The hypoxic conditions persist until the vascularity increases to match the cellularity. Thephysiological role of oxygen in osseous repair is presented. The present technique as well as the previously published microelectrode technique have demonstrated bone formation in vivo can take place under hypoxic conditions but the mechanism is not known and requires further investigation.