A PTHrP Gradient Drives Mandibular Condylar Chondrogenesis via Runx2.

The mandibular condylar cartilage (MCC) is an essential component of the temporomandibular joint, which orchestrates the vertical growth of the mandibular ramus through endochondral ossification with distinctive modes of cell differentiation. Parathyroid hormone-related protein (PTHrP) is a master regulator of chondrogenesis; in the long bone epiphyseal growth plate, PTHrP expressed by resting zone chondrocytes promotes chondrocyte proliferation in the adjacent layer. However, how PTHrP regulates chondrogenesis in the MCC remains largely unclear. In this study, we used a Pthrp-mCherry knock-in reporter strain to map the localization of PTHrP+ cells in the MCC and define the function of PTHrP in the growing mandibular condyle. In the postnatal MCC of PthrpmCherry/+ mice, PTHrP-mCherry was specifically expressed by cells in the superficial layer immediately adjacent to RUNX2-expressing cells in the polymorphic layer. PTHrP ligands diffused across the polymorphic and chondrocyte layers where its cognate receptor PTH1R was abundantly expressed. We further analyzed the mandibular condyle of PthrpmCherry/mCherry mice lacking functional PTHrP protein (PTHrP-KO). At embryonic day (E) 18.5, the condylar process and MCC were significantly truncated in the PTHrP-KO mandible, which was associated with a significant reduction in cell proliferation across the polymorphic layer and a loss of SOX9+ cells in the chondrocyte layers. The PTHrP-KO MCC showed a transient increase in the number of Col10a1+ hypertrophic chondrocytes at E15.5, followed by a significant loss of these cells at E18.5, indicating that superficial layer-derived PTHrP prevents premature chondrocyte exhaustion in the MCC. The expression of Runx2, but not Sp7, was significantly reduced in the polymorphic layer of the PTHrP-KO MCC. Therefore, PTHrP released from cells in the superficial layer directly acts on cells in the polymorphic layer to promote proliferation of chondrocyte precursor cells and prevent their premature differentiation by maintaining Runx2 expression, revealing a unique PTHrP gradient-directed mechanism that regulates MCC chondrogenesis.

Mesenchymal Progenitor Regulation of Tooth Eruption: A View from PTHrP.

Tooth eruption is a unique biological process by which highly mineralized tissues emerge into the outer world, and it occurs concomitantly with tooth root formation. These 2 processes have been considered independent phenomena; however, recent studies support the theory that they are indeed intertwined. Dental mesenchymal progenitor cells in the dental follicle lie at the heart of the coupling of these 2 processes, providing a source for diverse mesenchymal cells that support formation of the highly functional tooth root and the periodontal attachment apparatus, while facilitating formation of osteoclasts. These cells are regulated by autocrine signaling by parathyroid hormone-related protein (PTHrP) and its parathyroid hormone/PTHrP receptor PPR. This PTHrP-PPR signaling appears to crosstalk with other signaling pathways and regulates proper cell fates of mesenchymal progenitor cell populations. Disruption of this autocrine PTHrP-PPR signaling in these cells leads to defective formation of the periodontal attachment apparatus, tooth root malformation, and failure of tooth eruption in molars, which essentially recapitulate primary failure of eruption in humans, a rare genetic disorder exclusively affecting tooth eruption. Diversity and distinct functionality of these mesenchymal progenitor cell populations that regulate tooth eruption and tooth root formation are beginning to be unraveled.

Msx2 Marks Spatially Restricted Populations of Mesenchymal Precursors.

Craniofacial development requires a set of patterning codes that define the identities of postmigratory mesenchymal cells in a region-specific manner, in which locally expressed morphogens, including fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs), provide instructive cues. Msx2, a bona fide target of BMP signaling, is a transcription factor regulating Runx2 and osterix (Osx), whose mutations are associated with cranial deformities in humans. Here we show that Msx2 defines osteo-chondro precursor cells in specific regions of the craniofacial mesenchyme at the postmigratory stage, particularly in the mandibular process and the posterior cranial vault. Analysis of Msx2-creER mice revealed that early mesenchymal cells in proximity to the BMP4-expressing mesenchyme were marked upon tamoxifen injection, and their descendants contributed to diverse types of mesenchymal cells in the later stage, such as chondrocytes and perichondrial cells of the transient cartilage, as well as osteoblasts and suture mesenchymal cells. By contrast, Osx-creER marked osteoblast precursors at the later stage, and their descendants continued to become osteoblasts well into the postnatal stage. Therefore, Msx2 marks spatially restricted populations of mesenchymal precursor cells with diverse differentiation potential, suggesting that extrinsic molecular cues can dictate the nature of postmigratory mesenchymal cells in craniofacial development.

Diverse contribution of Col2a1-expressing cells to the craniofacial skeletal cell lineages.

OBJECTIVES: Craniofacial skeletal development requires deliberate coordination of two distinct mechanisms of endochondral and intramembranous ossification. Col2a1-expressing cells encompass growth-associated skeletal progenitors in endochondral bones of the limb. The objective of this study was to determine the contribution of Col2a1-expressing cells to the craniofacial skeletal cell lineages. We hypothesize that Col2a1-expressing progenitors significantly contribute to various modes of ossification associated with the craniofacial development. METHODS: Cellular fates of Col2a1-expressing cells were studied based on a cre-loxP system using a Col2a1-cre transgene and an R26R-tdTomato reporter allele. We analysed three distinct locations of the craniofacial skeletal complex representing unique ossification mechanisms: the cranial base, the calvaria and the mandibular condyle. RESULTS: Col2a1-cre consistently marked a majority of skeletal cells in the cranial base. Interestingly, Col2a1-cre also marked a large number of osteoblasts and suture mesenchymal cells in the calvaria, in addition to chondrocytes in the underlying transient cartilage. In the mandibular condyle, Col2a1-cre marked chondrocytes and osteoblasts only during the growth phase. CONCLUSIONS: Col2a1 is expressed by progenitors of the skeletal lineage in canonical endochondral bone formation occurring in the cranial base. In contrast, other ossification mechanisms of the craniofacial complex utilize Col2a1-expressing cells in a different manner, whereby Col2a1 may be expressed in more differentiated or transient cell types of the skeletal lineage.

The fate of Osterix-expressing mesenchymal cells in dental root formation and maintenance.

OBJECTIVES: Osterix (Osx)-expressing mesenchymal cells are progenitors for tooth root forming cells. The aim of this study was to reveal the fates of Osx-expressing cells during and after root formation using a lineage tracing experiment. MATERIAL AND METHODS: To reveal the fates of Osx-expressing dental mesenchymal progenitors, we took advantage of tamoxifen-inducible Cre reporter system. Osx-creER; R26R-tdTomato mice received tamoxifen (0.1 mg/body) at postnatal day 3 (P3). In this system, Osx-expressing at P3 (Osx-P3) cells undergo recombination, and they and their descendants continue to express Tomato red fluorescence protein permanently. Mandibles were dissected at serial time points ranging from P4 to P116 to investigate how Osx-P3 cells participated in root formation. Tomato+ cells on frozen sections were imaged under fluorescence microscopy. RESULTS: Osx-P3 cells and their descendants differentiated into all kinds of cells that contributed to the root and periodontal tissues, such as odontoblasts, cementoblasts, alveolar bone osteoblasts and periodontal ligament (PDL) cells during root formation. Even after root formation was completed, they persisted in dental pulp and PDL to provide progenitor cells for odontoblasts and cementoblasts. CONCLUSION: Osx-expressing cells play important roles in the entire processes of tooth root formation; their progeny continue to contribute to maintenance of tooth root even after root formation is complete.

Post-Fukushima radiation education for public health nursing students: a case study.

BACKGROUND: The recent Fukushima Nuclear Power Plant accident was one of more than 200 serious nuclear/radiation incidents (accidents and disasters) that occurred worldwide since 1945. The current Fukushima disaster is in the recovery phase with the decreasing levels of radiation in the environment. However, fears and stigma related to the perceived risk of radiation exposure persist among the general population. INTRODUCTION: To improve on students' preparedness for social and public health challenges after a radiation incidence, radiation education was provided for undergraduate public health nursing students. AIM: This case study reports the development and implementation of the first class of radiation education in public health nursing, as well as students' reflections on their class experience. METHODS: We included a 90-min radiation class in an undergraduate public health nursing course in Tokyo, Japan. Lectures/discussion on technical and environmental aspects provided the minimally essential content for basic radiation knowledge. After class, all the 65 students were invited to freely write their reflections on the class. With their consent, 61 students' anonymous written accounts were qualitatively analysed. RESULTS: Five themes emerged: awareness of ignorance about radiation, problems produced by the mass media, becoming knowledgeable about radiation, public health nurses' role, and trustful and enjoyable lecture. DISCUSSION: The class inspired students to consider social, psychological and relational aspects of knowing and not knowing about radiation and their future professional role. CONCLUSION AND IMPLICATIONS FOR NURSING: Once radiation is taught at school, nursing students will emerge as professionals with the belief that radiation is within their professional purview. Education is key to disaster prevention, preparation, response and recovery. Given the ubiquitous nature of health challenges after a radiation incident, radiation education is indispensable for nursing students worldwide.

The performance of a novel ball-tipped Flush knife for endoscopic submucosal dissection: a case-control study.

BACKGROUND: Endoscopic submucosal dissection (ESD) using short needle knives is safe and effective, but bleeding is a problem due to low haemostatic capability. AIM: To assess the performance of a novel ball-tipped needle knife (Flush knife-BT) for ESD with particular emphasis on haemostasis. METHODS: A case-control study to compare the performance for ESD of 30 pairs of consecutive early gastrointestinal lesions (oesophagus: 12, stomach: 32, colorectum: 16) with standard Flush knife (F) vs. Flush knife-BT (BT). Primary outcome was efficacy of intraprocedure haemostasis. Secondary outcomes included procedure time, procedure speed (dividing procedure time into the area of resected specimen), en bloc resection rate and recurrence rate. RESULTS: Median intraoperative bleeding points and bleeding points requiring haemostatic forceps were smaller in the BT group than in the F group (4 vs. 8, P < 0.0001, 0 vs. 3, P < 0.0001). There was no difference between groups for procedure time; however, procedure speed was shorter in the BT group (P = 0.0078). En bloc and en bloc R0 resection rates were 100%, with no perforation or post-operative bleeding. No recurrence was observed in either group at follow-up 1 year postprocedure. CONCLUSIONS: Ball-tipped Flush knife (Flush knife-BT) appears to improve haemostatic efficacy and dissection speed compared with standard Flush knife.

Retrospective study of technical aspects and complications of endoscopic submucosal dissection for laterally spreading tumors of the colorectum.

BACKGROUND AND STUDY AIMS: Laterally spreading tumors - non granular type (LST-NG) are more often considered candidates for endoscopic submucosal dissection (ESD) than laterally spreading tumors - granular type (LST-G), because of their higher potential for submucosal invasion. However, ESD for LST-NG can be technically difficult. The aim of our study was to compare our ESD results for LST-NG and for LST-G. PATIENTS AND METHODS: Ninety-nine LST-NG and 169 LST-G measuring 20 mm in size or more were removed by ESD. We retrospectively evaluated the clinicopathological features of the tumors and treatment results (en bloc resection rate, procedure time and speed, rate of use of ancillary devices, and complication and recurrence rates). RESULTS: Histopathology revealed that there were more submucosally invasive lesions in the LST-NG than in the LST-G group (28 % vs. 9 %; P < 0.0001). The en bloc resection rate, en bloc R0 resection rate, and en bloc curative resection rate of LST-NG were similar to those of LST-G (LST-NG: 99 %, 98 %, and 88 %; LST-G: 99 %, 98 %, and 91 %). In LST-NG, the median procedure time tended to be longer (LST-NG: 69 min; LST-G: 60 min) and the median procedure speed was slower (LST-NG: 0.15 cm (2)/min; LST-G: 0.25 cm (2)/min; P < 0.0001). Use of ancillary devices was higher for LST-NG (38 % vs. 15 % for LST-G; P < 0.0001), as was the perforation rate (5.1 % vs. 0.6 % for LST-G; P = 0.027). No recurrence was seen in either group. CONCLUSIONS: ESD was an effective treatment method for both LST-NG and LST-G. However, the degree of technical difficulty appears higher for LST-NG than for LST-G lesions, as shown by the lower dissection speed and higher perforation rate. ESD for LST-NG should probably be performed by those with significant experience of colorectal ESD.

Analysis of calcium deposits in calcific periarthritis.

OBJECTIVE: To determine if hydroxyapatite (HAP), octacalcuim phosphate (OCP), or tricalcium phosphate (TCP) can be found in the calcium deposits in calcific periarthritis. METHODS: Thirty-six specimens from 34 patients who had acute inflammation and roentgenographically recognized calcification in soft tissue were analyzed. Twenty-three patients with calcific tendinitis in the shoulder and 11 with calcific periarthritis at other sites were included. We prepared 2 kinds of samples from each specimen; a dried sample (washed and dried calcific deposit), and a sample heated to 1,000 degrees C. All were analyzed by X-ray diffraction, Raman spectroscopy, infrared absorption spectroscopy, and X-ray fluorescence spectrometry for calcium and phosphorus molar ratio. Synthetic HAP was used as the control in each analysis. RESULTS: The X-ray diffraction patterns of all dried samples were similar to those of HAP and carbonate apatite. We found no diffraction patterns of OCP or TCP. However, an OH- group at 3570cm(-1) was observed with Raman spectroscopy for samples heated to 1,000 degrees C and synthetic HAP, but not for the dried samples. Infrared absorption spectroscopy also confirmed an OH- group for samples heated to 1,000 degrees C and synthetic HAP, and confirmed that dried samples contained carbonate. CONCLUSION: Calcium deposits are composed of carbonate apatite. HAP, OCP, and TCP were not identified in any deposits.

Nondiffuse fatty change of the liver: discerning pseudotumor on MR images enhanced with ferumoxides-initial observations.

PURPOSE: To clarify the findings of nondiffuse fatty change of the liver on ferumoxides-enhanced magnetic resonance (MR) images. MATERIALS AND METHODS: Of 202 patients who underwent ferumoxides-enhanced MR imaging, eight who had nondiffuse fatty change of the liver at computed tomography (CT) were examined as study subjects. MR imaging findings before and 1 hour after ferumoxides administration were compared with CT findings. RESULTS: Focal fatty areas of the liver showing low attenuation on CT images were depicted as areas of relatively high intensity on the ferumoxides-enhanced T1-weighted images in all patients. On enhanced T2-weighted images, focal fatty change showed relatively high intensity in three and isointensity in one of the four patients. Focal spared areas appearing as areas of relatively high attenuation on CT images were depicted as areas of relatively low intensity on the ferumoxides-enhanced T1- and T2-weighted images in all patients. CONCLUSION: Although prior reports of hepatic MR imaging with ferumoxides indicated that there is accumulation of ferumoxides within focal fatty areas that are no longer seen after the administration of contrast medium, this study revealed that focal fatty change and focal spared areas of fatty liver may be pseudotumors because of the relatively high intensity of fatty areas of the liver. Radiologists can distinguish these conditions from hepatic tumors by using the opposed-phase gradient-echo sequence or the fat-saturation technique.

Serum nucleotide pyrophosphohydrolase activity; elevated levels in osteoarthritis, calcium pyrophosphate crystal deposition disease, scleroderma, and fibromyalgia.

OBJECTIVE: Quantification of serum nucleotide pyrophosphohydrolase (NTPPHase) activity in healthy subjects and in patients with various rheumatic diseases or with quad/hemiplegia, hemodialysis, or renal transplant. METHODS: Colorimetric assay of enzyme activity in serum. RESULTS: Serum NTPPHase activity in 85 healthy subjects was independent of age or sex and was highly reproducible in each individual. The biologic and methodologic coefficients of variation were nearly identical. Elevated enzyme levels were found in sera from patients with osteoarthritis/spondylosis, calcium pyrophosphate dihydrate (CPPD) crystal deposition, scleroderma, fibromyalgia, or hemodialysis. Renal transplant patients receiving cyclosporine had the highest enzyme activity of any group, whereas transplant patients not taking this drug had normal levels. Histograms of values in all groups showed a normal distribution. CONCLUSION: Serum NTPPHase activity levels were significantly elevated in patients with degenerative arthritis whether or not CPPD crystals were present, in patients with either scleroderma or fibromyalgia, and in patients receiving hemodialysis therapy or taking cyclosporine.

Optimal dose of hepatobiliary contrast agent for MR cholangiography: experimental study in rats.

The purpose of this study is to clarify the optimal dose of gadolinium-ethoxybenzyl-diethylenetriaminepentaacetic acid (Gd-EOB-DTPA) for cholangiography in conventional T1-weighted imaging. We divided 30 rats into three dose groups (3, 10, and 30 micromol/kg). For the in vitro study, we collected bile and measured the concentration of gadolinium in bile after Gd-EOB-DTPA injection. T1-weighted images of the collected bile were obtained for measurement of signal intensity. For the in vivo study, we obtained T1-weighted images before and after injection and evaluated bile duct/liver contrast by the signal intensity ratio and visual assessment of the images. The gadolinium concentration had an early peak; however, the signal intensity of the bile had a later peak because of the high gadolinium concentration during the early phase, which induced a T2-shortening effect. Optimal bile duct/liver contrast was obtained in the 10-micromol/kg groups at all time points. We conclude that the optimal dose of Gd-EOB-DTPA for MR cholangiography in rats is 10 micromol/kg, one-third of the dose used in liver imaging.

Nucleotide pyrophosphohydrolase in human synovial fluid.

OBJECTIVE: To identify the molecular forms of ectonucleotide pyrophosphohydrolase (NTPPHase) in human synovial fluid (SF). METHODS: We examined synovial fluids from 32 patients with various joint diseases [10 calcium pyrophosphate dihydrate (CPPD) deposition disease; 7 osteoarthritis (OA); 6 rheumatoid arthritis (RA); 3 after total knee arthroplasty (TKA); 6 olecranon bursa] and 3 normal joint fluids. Joint fluids were analyzed after sequential centrifugation for NTPPHase activity and by Western blot using polyclonal antibodies against 127 kDa porcine articular cartilage vesicle-associated NTPPHase and against PC-1 and 58 kDa, 2 other ecto-NTPPHases. Lysate from human synoviocytes, porcine chondrocytes, and their conditioned media were examined using antibodies to these ecto-NTPPHases. Radiographs of joints from which fluid was obtained were graded for degenerative changes 0-4 using a standard method. RESULTS: NTPPHase activity was found in all pathological and normal SF tested and correlated with the degree of radiographic degeneration (r = 0.55, p < 0.05). NTPPHase specific activity in ultracentrifugation pellets was highest in CPPD deposition disease fluids (p < 0.05). 127 kDa enzyme was found in both sedimentable and soluble fractions from CPPD, OA, TKA, and normal fluids, and was extensively degraded in all inflammatory fluids. Intact 115 kDa PC-1 was found only in the 2 CPPD fluids with the highest NTPPHase activity. 58 kDa enzyme was found in most fluids, predominantly in the soluble fraction. 127 kDa protein was identified in human synoviocyte conditioned media but not in cell lysate, while PC-1 and 58 kDa proteins were found in the cell lysate but not in the conditioned media. CONCLUSION: There was no disease specific association with any one ecto-NTPPHase. Total enzyme activity correlated with the degree of degenerative change. The specific activity of pelletable 127 kDa enzyme was higher in fluids containing CPPD crystals. All 3 ecto-NTPPHases or their presumed degradation products were detectable in some pathologic and normal fluids. A 200 kDa reactive band often accompanied reactivity to the 127 kDa enzyme. PC-1 and 127 kDa proteins were extensively degraded in inflammatory SF, while 58 kDa protein was not. The relative contribution of each of these enzymes to inorganic pyrophosphate production by human joint tissues remains unclear.

MR cholangiopancreatography and MR urography: improved enhancement with a negative oral contrast agent.

At magnetic resonance (MR) cholangiopancreatography and MR urography with an oral negative contrast agent, low signal intensity was produced in phantoms. In 20 patients suspected of having biliary tract and pancreatic diseases and in 20 healthy volunteers, the signal intensity in the gastrointestinal tract was almost completely eliminated with the negative contrast agent. Differences in image quality between pre- and postcontrast images were statistically significant.

[Calcification of cervical ligamentum flavum--analysis of calcium compounds and histopathological findings].

We operated 3 patients with cervical myelopathy due to calcification of ligamentum flavum (CLF). Specimens collected surgically were subjected to X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy (Raman) analysis and also histopathological examination. The calcium compounds deposited were calcium pyrophosphate dihydrate (CPPD) in Case 1, apatite in Case 2, and a double-layer structure with an outer CPPD layer and an inner carbonate apatite layer in Case 3. Histopathologically, CPPD deposition in Case 1 could be distinguished from apatite deposition in Case 2 and 3 by hematoxylin-eosin stain. Chondrocytes were observed in all 3 cases, suggesting the chondrocytes may have played a role in calcification in these three cases. To date, 62 cases of CLF (including the present cases) have been reported, and analysis of calcium compound has been performed for 29 of them. Of these 29, 15 were analyzed as calcium phosphate compounds, 9 as CPPD and 4 cases as mixed crystals like Case 3. However the analysis method and result about CPPD are no problem, the analysis result of calcium phosphate compounds depends on the using methods. Calcium phosphate or undetectable compounds was identified by XRD as hydroxyapatite (HAp), and by FTIR as calcium phosphate or undetectable compound. Analysis of calcium phosphate compounds should be condacted and identified by XRD, FTIR, and Raman. We propose two possible mechanisms for the formation of the double-layer structure in Case 3: one is formation of apatite first followed by deposition of CPPD outside, and the other is formation of CPPD first followed by conversion of CPPD in the central region to apatite. What the process of formation of the double-layer was in this cases remains unclear.

Identification of a nucleotide pyrophosphohydrolase from articular tissues in human serum.

OBJECTIVE: To characterize the nucleotide pyrophosphohydrolase (NTPPHase) in human serum. METHODS: NTPPHase activity and kinetic analysis were performed using thymidine monophosphate paranitrophenyl ester (TMPNP) or 32Pgamma-labeled ATP as substrate. Sera were chromatographed (dye column), and peak fractions were analyzed kinetically and by immunoblot using antibodies to 127-kd articular cartilage vesicle (ACV) NTPPHase as well as to PC-1 and to 58 kd, two plasma membrane ecto-NTPPHases. Enzyme activity was measured before and after sample ultracentrifugation. RESULTS: NTPPHase activity was found in all sera tested (2 normal subjects, 9 arthritis patients). Specific activity was increased 9-32-fold after chromatography; 60-80% of total activity was recovered in a single peak containing an approximately 100-kd soluble peptide related to the 127-kd ACV enzyme. The apparent Km of this peptide (TMPNP) was virtually identical to that of the porcine ACV 127-kd enzyme. No immunoreactivity against PC-1 or 58-kd NTPPHase was found. CONCLUSION: Human serum NTPPHase is derived from 127-kd ACV-related enzyme.