A novel function for lysyl oxidase in pluripotent mesenchymal cell proliferation and relevance to inflammation-associated osteopenia.

Lysyl oxidase is a multifunctional enzyme required for collagen biosynthesis. Various growth factors regulate lysyl oxidase during osteoblast differentiation, subject to modulation by cytokines such as TNF-α in inflammatory osteopenic disorders including diabetic bone disease. Canonical Wnt signaling promotes osteoblast development. Here we investigated the effect of Wnt3a and TNF-α on lysyl oxidase expression in pluripotent C3H10T1/2 cells, bone marrow stromal cells, and committed osteoblasts. Lysyl oxidase was up-regulated by a transcriptional mechanism 3-fold in C3H10T1/2 cells, and 2.5-fold in bone marrow stromal cells. A putative functional TCF/LEF element was identified in the lysyl oxidase promoter. Interestingly, lysyl oxidase was not up-regulated in committed primary rat calvarial- or MC3T3-E1 osteoblasts. TNF-α down-regulated lysyl oxidase both in Wnt3a-treated and in non-treated C3H10T1/2 cells by a post-transcriptional mechanism mediated by miR203. Non-differentiated cells do not produce a collagen matrix; thus, a novel biological role for lysyl oxidase in pluripotent cells was investigated. Lysyl oxidase shRNAs effectively silenced lysyl oxidase expression, and suppressed the growth of C3H10T1/2 cells by 50%, and blocked osteoblast differentiation. We propose that interference with lysyl oxidase expression under excess inflammatory conditions such as those that occur in diabetes, osteoporosis, or rheumatoid arthritis can result in a diminished pool of pluripotent cells which ultimately contributes to osteopenia.

Inhibition of lysophosphatidic acid receptor-2 expression by RNA interference decreases lysophosphatidic acid-induced urokinase plasminogen activator activation, cell invasion, and migration in ovarian cancer SKOV-3 cells.

AIM: To explore the role of lysophosphatidic acid receptor-2 (LPA2) in regulating lysophosphatidic acid (LPA)-induced urokinase plasminogen activator (uPA) activation, cell invasion, and migration in human ovarian cancer cell line SKOV-3. METHODS: SKOV-3 cells were stimulated with LPA. Cell supernatant uPA level and activity were measured using enzyme-linked immunosorbent assay. LPA2 mRNA expression was inhibited with LPA2-specific small interfering RNA (siRNA) and examined using semiquantitative reverse transcriptase-polymerase chain reaction. LPA-induced cell invasion and migration in transfected cells were evaluated by a Matrigel invasion chamber and a Transwell chemotaxis chamber, respectively. RESULTS: LPA stimulation significantly enhanced in vitro uPA activity in time- and dose-dependent manner. The levels of LPA-induced uPA protein decreased by 55% in LPA2 siRNA-transfected cells compared with negatively transfected cells at 24 hours after being treated with 80 micromol/L LPA (0.75+/-0.03 vs 0.34+/-0.04, P=0.004). In the LPA2 specific siRNA-transfected SKOV-3 cells, LPA treatment at 80 micromol/L induced considerably less invasion and migration compared with negative control siRNA-transfected SKOV-3 cells (invasion: 178+/-17.2 vs 36.2+/-3.3, P=0.009; migration: 220.4+/-25.5 vs 57+/-7.6, P=0.009). CONCLUSION: LPA2 has an essential role in LPA-induced uPA activation and tumor cell invasion in ovarian cancer SKOV-3 cells.

Accurately shaped tooth bud cell-derived mineralized tissue formation on silk scaffolds.

Based on the successful use of silk scaffolds in bone tissue engineering, we examined their utility for mineralized dental tissue engineering. Four types of hexafluoroisopropanol (HFIP) silk scaffolds-(250 and 550 microm diameter pores, with or without arginine-glycine-aspartic acid (RGD) peptide) were seeded with cultured 4-day postnatal rat tooth bud cells and grown in the rat omentum for 20 weeks. Analyses of harvested implants revealed the formation of bioengineered mineralized tissue that was most robust in 550 microm pore RGD-containing scaffolds and least robust in 250 microm pore sized scaffolds without RGD. The size and shape of the silk scaffold pores appeared to guide mineralized tissue formation, as revealed using polarized light imaging of collagen fiber alignment along the scaffold surfaces. This study is the first to characterize bioengineered tissues generated from tooth bud cells seeded onto silk scaffolds and indicates that silk scaffolds may be useful in forming mineralized osteodentin of specified sizes and shapes.

Effect of neurotrophins on differentiation, calcification and proliferation in cultures of human pulp cells.

Neurotrophins (NTs) are expressed during tooth development. However, little is known about a role of NTs in differentiation of pulp cells into mineralizing cells. In this study, mRNA expressions of hard tissue-related proteins, calcification and proliferation are examined in cultures of human pulp (HP) cells. Nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin (NT)-3 and NT-4/5 increased the mRNA levels of dentin sialophsphoprotein, alkaline phosphatase, osteopontin, type I collagen and bone morphogenetic protein-2 and mineral deposition in cultures of HP cells. The increased levels and manners varied, depending on the concentrations of NTs and hard-tissue related protein tested. On the other hand, only NGF significantly stimulated DNA synthesis in cultures of HP cells. These findings suggest that NTs characteristically regulate hard-tissue related protein expression, calcification and proliferation in pulp cells. NTs may accelerate pulp cell differentiation.

[Intra-operative fluoroscopy for detecting pedicle screw violation in lumbar vertebrae by guided pin axial position].

OBJECTIVES: To develop an applicable and accurate intra-operative fluoroscopical angle to observe the placement of pedicle screw and pedicle violation. METHODS: Lateral radiographs were taken on eighty lumbar pedicles in eight cadaveric vertebral specimens to measure the sagittal pedicle angles. The pedicles of the vertebral arch were scanned by computed tomography (CT) to measure the transverse pedicle angles. Guided pins and pedicle screws were inserted by awl into the pedicles through the holes drilled in the lumbar pedicles so as to establish 3 types of model: medial violation (n = 29), lateral violation (n = 25), and properly placed screw (n = 26) the anterior and posterior portions of the lumbar pedicle cortex could be identified by the fluoroscopy C-arm. Fluoroscopy with the aid of C-arm was taken on each pedicle in five different directions: lateral position; anteroposterior position; pedicle axial position; guided pin axial position; and pedicle screw axial position. The positions of guided pin and pedicle screw were evaluated. The gold standard was acquired by excising the vertebral plate and observing directly. RESULTS: Nine cases of medial violation were discovered and the accuracy rate was 33.75% by anteroposterior fluoroscopy. The accuracy rate was 95% by the pedicle axial fluoroscopes, including 4 cases of medial inclination but properly placed screws were mistaken for medial violation by pedicle axial fluoroscope. The accuracy rate was 100% by guided pin axial position fluoroscopy, including 29 cases of medial violations, 25 cases of lateral violation, and 26 cases of properly placed screws. CONCLUSION: The approach of intra-operative fluoroscopy of guided pin axial position is a reliable technology for detecting pedicle violation with the aid of C-arm. The incidence of flexible pedicle screw from human factors can be reduced.

Promotion of functioning of human periodontal ligament cells and human endothelial cells by nerve growth factor.

BACKGROUND: We have previously shown that cultured human periodontal ligament (HPL) cells produce nerve growth factor (NGF) and express mRNA of tyrosine kinase receptor (trkA), a high-affinity receptor of NGF. These findings suggest that NGF modulates the differentiation and proliferation of the periodontal ligament cells by paracrine and autocrine functions in vivo. Endothelial cells also express NGF and trkA. Therefore, NGF may regulate functions of periodontal ligament cells and endothelial cells during periodontal tissue regeneration. METHODS: Effects of NGF on expressions of bone/cementum-related proteins (osteocalcin [OC], bone sialoprotein [BSP], bone morphogenetic protein [BMP-7], core binding factor alpha [Cbfa-1], and type I collagen), calcification in HPL cells, and proliferation and mRNA expression of vascular endothelial growth factor (VEGF), an endothelial cell mitogen, in human microvascular endothelial cells (HMVECs) were examined. RESULTS: NGF elevated mRNA levels of OC, BSP, BMP-7, Cbfa-1, and type I collagen and enhanced mineral deposition in cultures of HPL cells. Furthermore, NGF stimulated mRNA expressions of VEGF-A and VEGF-B and cell proliferation in HMVEC. CONCLUSION: These findings suggest that the functional regulation of periodontal ligament cells and endothelial cells by NGF might result in the acceleration of periodontal tissue regeneration in vivo.

Effect of bone morphogenetic proteins-4, -5 and -6 on DNA synthesis and expression of bone-related proteins in cultured human periodontal ligament cells.

Bone morphogenetic proteins (BMPs) have multiple functions in the development and growth of skeletal and extraskeletal tissues. Therefore, BMPs may regulate the regeneration of periodontal tissue. To investigate this issue, we examined the effects of BMP-4, -5 and -6 on DNA synthesis and the expression of bone-related proteins in cultures of human periodontal ligament (HPL) cells. The expression of bone-related proteins was determined by Real-time polymerase chain reaction and enzyme linked immunosorbent assay in cultures of HPL cells. DNA synthesis was estimated by measuring bromoderoxyuridine incorporation. It was found that BMP-4, -5 and -6 enhanced DNA synthesis dose-dependently. BMP-4 and -5 increased the levels of osteopontin, BMP-2, alkaline phosphatase and core binding factor alpha 1 mRNAs. BMP-6 stimulated the expression of osteopontin, BMP-2, ALPase and osteoprotegerin. These findings show that BMP-4, -5 and -6 have different actions on the expression of bone-related proteins and may play a role in the regeneration of periodontal tissue by promoting cell proliferation and protein expression.

[The surgical management of sacral tumors].

OBJECTIVE: To investigate the way of sacral tumors surgical treatment. METHODS: This retrospective study included 119 cases of sacral tumors surgically treated from July, 1996 to December, 2001. The age of patients ranged from 18 to 80 years (mean 57 years), including of 72 male and 47 female. Out of the patients, there were 52 chordomas, 16 giant cell tumor, 5 neurofibroma, 23 metastases tumors, 9 myeloma, 2 osteoblastomas, 5 aneurysmal bone cysts, 3 osteosarcomas, 4 chondrosarcomas. Posterior approach and combined anterior-posterior approach were used in 83 and 36 cases respectively. Twenty-nine patients had received surgical management at least once and 16 of them had received radiation therapy before came to our department. RESULTS: Three patients died on the complication around the surgery. Most of the patients with metastases tumor or multiple myeloma died 1 to 3 years after the surgery. Out of three osteosarcoma patients, 2 died and one alive with tumor. Three chondrosarcoma patients died, and one alive with tumor. Out of 52 chordoma patients, 3 patients had died of metastatic chordoma, 3 patients died of many times recurrence. Among the other 46 patients who were stay alive, 31 were free from disease with average follow-up time of 42 months. In the patients whose sacral nerve roots had been reserved bilaterally at and above S(3) level, the sphincter muscle function of bladder and bowl was good. While the function of sphincter muscle impaired in 2 patients with nerve roots reserved only at and above S(1) level. To manage these 2 patients, indwelling bladder catheters were used, but colostomy had not been performed. CONCLUSIONS: Complete resection of tumor (radical surgery when possible) is the most effective way to manage sacral tumors. Postoperative adjuvant radiation therapy can reduce the tumor recurrence rate, but it also can cause troubles that would hinder further surgical managements. Even if the tumor is relatively huge and the upper resection margin is as high as at S(1) or S(2) level, the tumor can be removed successfully by posterior approach and the postoperative complications could be accepted. To the patients with aneurysmal cyst or giant cell tumor on sacrum, for control bleeding purpose, anterior approach should be performed to ligate the bilateral internal iliac artery.