The Quantitative Assessment of Osteoinductivity of Human Demineralized Bone Matrix and cDNA Array Analysis of Osteogenic Differentiation in Human Periosteal Cells

Demineralized bone matrix (DBM) has been extensively utilized in orthopeadic, periodontal, and maxillofacial applications. DBM comprises bone morphogenetic proteins (BMPs) that are essential regulators for endochondral bone formation. The current study was undertaken to establish the quantitative in vitro assays for determining the osteoinductive potential of human DBM and the use of cDNA gene expression array technology for examining gene expression profiles during osteoblast differentiation in human periosteal cells. BMP-4 is one of the most osteoinductive factors and has been investigated for clinical applications. For this reason, BMP-4 was designated as the osteoinductive protein for use in the quantitative in vitro assay. At the same time, the osteoinductivity of DBM was measured by histomorphometric analysis of new bone formation in an in vivo athymic mouse bioassay. DBM exhibiting high osteoinductivity in the nude mouse bioassay contained higher amounts of extractable BMP-4 than did DBM samples possessing low osteoinductivity. In the study of the effect of residual calcium on extractability of BMP-4, the extractable BMP-4 content was undetectable in nondemineralized bone matrix. As the residual calcium level decreased, the extractability of BMP-4 in DBM increased. DBM particles less than 250 microns yielded the lowest amounts of extractable BMP-4 among all size groups tested. Bone particles in the 500–710 micron size range provided for DBM with the highest extractability of BMP-4. In the donor age study, the BMP-4 content in the protein extracts of DBM appears to be age-dependent, with DBM from younger donors being most likely to have greater BMP-4 quantity. In the in vitro dose-response studies, DBM-conditioned media (DBM-CM) enhanced alkaline phosphatase activities of human periosteal cells in a dose-dependent fashion. Alkaline phosphatase activities in both biochemical and histochemical analysis increased in the DBM-CM treated flasks compared to non-treated flasks after five days of incubation. In gene expression analysis of osteogenic differentiation using cDNA array analysis, a few differentially expressed genes, including biglycan, TGF-β1, and TGF-βR1 were up-regulated, whereas collagen14A1 was down-regulated in response to DBM-CM treatment. In the present study, gene expression array technology provides insight to the biological process of osteogenic differentiation

Relative telomere length and oxidative DNA damage in hypertrophic ligamentum flavum of lumbar spinal stenosis

Background Lumbar spinal stenosis (LSS) is a common cause of low back pain with degenerative spinal change in older adults. Telomeres are repetitive nucleoprotein DNA sequences of TTAGGG at the ends of chromosomes. Oxidative stress originates from an imbalance in pro-oxidant and antioxidant homeostasis that results in the production of reactive oxygen species (ROS). The purpose of this study was to investigate relative telomere length (RTL) and oxidative DNA damage in ligamentum flavum (LF) tissue from LSS patients. Methods Forty-eight patients with LSS participated in this study. Genomic DNA from non-hypertrophic and hypertrophic LF tissue were analyzed by real-time polymerase chain reaction for relative telomere length (RTL). 8-hydroxy 2′-deoxygaunosine (8-OHdG) levels were determined by using enzyme-linked immunosorbent assay. We cultivated LF fibroblast cells from patients in different ages (61, 66, and 77 years). After each cultivation cycle, we examined RTL and senescence-associated β-galactosidase (SA-β-gal) expression. Results The hypertrophic LF had significantly lower RTL than non-hypertrophic LF (P = 0.04). The levels of 8-OHdG were significantly higher in hypertrophic LF compared to non-hypertrophic LF (P = 0.02). With advancing cell culture passage, the number of cells in each passage was significantly lower in hypertrophic LF fibroblast cells than non-hypertrophic LF fibroblast cells. When evaluated with SA-β-gal staining, all senescent LF fibroblast cells were observed at earlier passages in hypertrophic LF compared with non-hypertrophic LF fibroblast cells. Discussion Our results showed that patients with LSS displayed an accelerated RTL shortening and high oxidative stress in hypertrophic LF. These findings implied that telomere shortening and oxidative stress may play roles in the pathogenesis of hypertrophic LF in lumbar spinal stenosis

Alu hypermethylation and high oxidative stress in patients with musculoskeletal tumors

Background Alu is one of the non-autonomous element retrotransposons, constituting nearly 11% of the human DNA. Methylation changes of the Alu element can cause genomic instability, a hallmark of cancer development, ultimately leading to the development of cancer. Epigenetic factors may induce the aberrant methylation of Alu and also oxidative stress. However, current knowledge of Alu methylation and oxidative stress is limited. There are few studies that have evaluated Alu methylation and oxidative stress on musculoskeletal tumor progression. Therefore, the present study evaluated the status of Alu methylation in musculoskeletal (MS) tumor, adjacent tissues, and blood leukocytes from MS tumor subjects, as well as unaffected participants. Moreover, we also investigated the oxidative stress status in MS tumor subjects and the control participants and determined the correlation between Alu methylation in MS tumors and that in blood leukocytes. Methods Musculoskeletal tumors from musculoskeletal tumor patients (n = 40) were compared to adjacent tissues (n = 40). The blood leukocytes from musculoskeletal tumor patients were compared to the blood leukocytes from controls (n = 107). Alu methylation status was analyzed using quantitative combined bisulfite restriction analysis (COBRA). In addition, 8–hydroxy 2′–deoxyguanosine (8–OHdG) values were determined using enzyme—linked immunosorbent assay. Results Alu methylation values in MS tumors were statistically significantly higher than those in adjacent tissues (P = 0.035). Similarly, Alu methylation statuses in the blood leukocytes of MS tumor subjects were statistically greater than those of control participants (P < 0.001). Moreover, there was a positive association between Alu methylation levels in MS tumors and blood leukocytes (r = 0.765, P < 0.001). In addition, the highest tertile was significantly associated with the risk of MS tumors (OR = 14.17, 95% CI [5.08–39.51]; P < 0.001). The 8-OHdG values in MS tumors were statistically higher than in adjacent tissues (P < 0.001) and circulating 8-OHdG levels were substantially greater in MS tumor subjects than in the control participants (P < 0.001). Discussion These findings suggest that Alu methylation in blood leukocytes and plasma 8-OHdG might represent non-invasive biomarkers to help diagnose MS tumors. Therefore, Alu hypermethylation and high oxidative stress might be involved in the pathogenesis of the musculoskeletal tumors

Serum adiponectin and transient elastography as non-invasive markers for postoperative biliary atresia

<p>Abstract</p> <p>Background</p> <p>Biliary atresia (BA) is a progressive inflammatory disorder of the extrahepatic bile ducts leading to the obliteration of bile flow. The purpose of this study was to determine serum adiponectin in BA patients and to investigate the relationship of adiponectin with clinical parameters and liver stiffness scores.</p> <p>Methods</p> <p>Sixty BA patients post Kasai operation and 20 controls were enrolled. The mean age of BA patients and controls was 9.6 ± 0.7 and 10.1 ± 0.7 years, respectively. BA patients were classified into two groups according to their serum total bilirubin (TB) levels (non-jaundice, TB < 2 mg/dl vs. jaundice, TB ≥ 2 mg/dl) and liver stiffness (insignificant fibrosis, liver stiffness < 7 kPa vs. significant fibrosis, liver stiffness ≥ 7 kPa). Serum adiponectin levels were analyzed by enzyme-linked immunosorbent assay. Liver stiffness scores were examined by transient elastography (FibroScan).</p> <p>Results</p> <p>BA patients had markedly higher serum adiponectin levels (15.5 ± 1.1 vs. 11.1 ± 1.1 μg/ml, <it>P </it>= 0.03) and liver stiffness than controls (30.1 ± 3.0 vs. 5.1 ± 0.5 kPa, <it>P </it>< 0.001). Serum adiponectin levels were significantly elevated in BA patients with jaundice compared with those without jaundice (24.4 ± 1.4 vs. 11.0 ± 0.7 μg/ml, <it>P </it>< 0.001). In addition, BA patients with significant liver fibrosis had remarkably greater serum adiponectin than insignificant fibrosis counterparts (17.7 ± 1.2 vs. 9.4 ± 1.1 μg/ml, <it>P </it>< 0.001). Subsequent analysis revealed that serum adiponectin was positively correlated with total bilirubin, hyaluronic acid, and liver stiffness (<it>r </it>= 0.58, <it>r </it>= 0.46, and <it>r </it>= 0.60, <it>P </it>< 0.001, respectively).</p> <p>Conclusions</p> <p>Serum adiponectin and liver stiffness values were higher in BA patients compared with normal participants. The elevated serum adiponectin levels also positively correlated with the degree of hepatic dysfunction and liver fibrosis. Accordingly, serum adiponectin and transient elastography could serve as the useful non-invasive biomarkers for monitoring the severity and progression in postoperative BA.</p

Isolation of Microvesicles from Human Circulating Neutrophils.

Neutrophil-derived microvesicles (NDMVs) are liberated by neutrophils upon cell activation by molecules. Once activated, neutrophils are primarily involved in acute inflammation; however, the microvesicles they produce are largely anti-inflammatory. NDMVs have been shown to protect cartilage during inflammatory arthritis. They exert these effects by inhibiting or affecting the function of target cells, including macrophages. NDMVs have the potential to act as disease-modifying agents, especially for inflammatory diseases. This protocol describes a method using differential centrifugation to separate neutrophils from whole human blood. Subsequently, neutrophils are identified by cytospin and Wright's staining, and then the NDMVs are isolated using differential centrifugation

Dickkopf-1 (Dkk-1) in plasma and synovial fluid is inversely correlated with radiographic severity of knee osteoarthritis patients

<p>Abstract</p> <p>Background</p> <p>Osteoarthritis (OA) is a common degenerative joint disease causing pain, stiffness, reduced motion, swelling, crepitus, and disability. Dickkopf-1 (Dkk-1) is a critical mediator of osteoblastogenesis and regulates the joint remodeling. The aim of this study was to examine plasma and synovial fluid Dkk-1 levels of patients with primary knee OA and to investigate their relationship with disease severity.</p> <p>Methods</p> <p>Thirty-five patients aged 55-83 years with knee OA and 15 healthy individuals were recruited into this study. Disease severity was determined using weight-bearing anteroposterior radiographs of the affected knee. The radiological grading of OA in the knee was performed according to the Kellgren-Lawrence grading system. Dkk-1 levels in both plasma and synovial fluid were evaluated using enzyme-linked immunosorbent assay.</p> <p>Results</p> <p>The average concentration of circulating Dkk-1 in the knee OA patients was remarkably lower than that of healthy controls (396.0 ± 258.8, 95%CI 307.1-484.9 vs 2348.8 ± 2051.5, 95%CI 1164.3-3533.3 pg/ml, p < 0.0001). Dkk-1 levels in synovial fluid were significantly lower than in paired plasma samples (58.6 ± 31.8, 95%CI 47.7-69.6 vs 396.0 ± 258.8, 95%CI 307.1-484.9 pg/ml, p < 0.001). Furthermore, both plasma and synovial fluid Dkk-1 levels were inversely correlated with radiographic severity (r = -0.78, p < 0.001 and r = -0.42, p = 0.01, respectively). Plasma Dkk-1 levels were also significantly correlated with synovial fluid Dkk-1 levels (r = 0.72, p < 0.001).</p> <p>Conclusions</p> <p>Dkk-1 levels in plasma and synovial fluid are inversely related to the severity of joint damage in knee OA. Dkk-1 could serve as a biochemical marker for determining disease severity and might play a potential role in the pathogenesis of the degenerative process of OA.</p

Role of Vitamin D in Osteoarthritis: Molecular, Cellular, and Clinical Perspectives

Osteoarthritis is a debilitating and degenerative disease which affects millions of people worldwide. The causes and mechanisms of osteoarthritis remain to be fully understood. Vitamin D has been hypothesised to play essential roles in a number of diseases including osteoarthritis. Many cell types within osteoarthritic joints appear to experience negative effects often at increased sensitivity to vitamin D. These findings contrast clinical research which has identified vitamin D deficiency to have a worryingly high prevalence among osteoarthritis patients. Randomised-controlled trial is considered to be the most rigorous way of determining the effects of vitamin D supplementation on the development of osteoarthritis. Studies into the effects of low vitamin D levels on pain and joint function have to date yielded controversial results. Due to the apparent conflicting effects of vitamin D in knee osteoarthritis, further research is required to fully elucidate its role in the development and progression of the disease as well as assess the efficacy and safety of vitamin D supplementation as a therapeutic strategy