Simon K.M. Lee

Effect Of Dexamethasone On Cultured Human Tenocytes And Its Reversibility By Platelet-derived Growth Factor

BACKGROUND Many cases of tendon rupture after glucocorticoid injections have been reported in the literature. Despite previous studies on the histological and biomechanical changes in tendons after glucocorticoid injections, the role of glucocorticoid in causing tendon rupture still remains controversial. The objective of this study was to determine whether glucocorticoid has deleterious effects on the cellular metabolism and collagen production of cultured human tenocytes and the reversibility of these effects by platelet-derived growth factor-BB (PDGFBB). METHODS Primary cultures of human tenocytes obtained from explants of healthy patellar tendon, harvested during anterior cruciate ligament reconstructions, were performed. The effects on cell viability, cell proliferation, and induction of apoptosis were measured by [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, 5-bromo-deoxyuridine incorporation, and DNA fragmentation assay, respectively. The effect on collagen synthesis was measured by (3) H-proline incorporation assay. RESULTS The number of viable cells was decreased, in a dose-dependent manner, by the administration of 10 (-9) to 10 (-4) -M dexamethasone. This dose range also suppressed cell proliferation. No apoptotic effect was detected. Treatment with 10 (-6) -M dexamethasone significantly reduced the amount of collagen synthesis. Co-incubation with 10 ng/mL of PDGFBB significantly reversed the effects caused by 10 (-6) -M dexamethasone. CONCLUSIONS Dexamethasone significantly decreased cell viability, suppressed cell proliferation, and reduced collagen synthesis in cultured human tenocytes. The effects were reversed by the simultaneous administration of PDGFBB.

Glucocorticoids suppress proteoglycan production by human tenocytes

Background The role of glucocortiocid injection therapy in spontaneous tendon rupture is controversial. We hypothesized that glucocorticoids suppress proteoglycan production in tendon and studied the in vitro effects of dexamethasone and triamcinolone on proteoglycan production by cultured human tenocytes. Material and methods We obtained primary cultures of human tenocytes from explants of healthy human patellar tendon. The human tenocytes were treated with 1 μM dexamethasone or 1 μM triamcinolone. The amount of proteoglycan production was measured by 35S-sulfate incorporation assay and compared with control cultures. The reversibility of the effect of dexamethasone by co-incubation with 10 ng platelet-derived growth factor (PDGFBB) was also tested. Results Treatment with 1 μM triamcinolone reduced the amount of 35S-sulfate incorporation to 80% of control cultures (p = 0.007), whereas 1 μM dexamethasone reduced it to 72% (p = 0.01). Co-incubation of 10 ng/mL PDGFBB with 1 μM dexamethasone returned the 35S-sulfate incorporation to a level thatwas significantly higher than for dexamethasone treatment alone (108%; p = 0.01). Interpretation Glucocorticoids suppressed proteoglycan production in cultured human tenocytes. The suppression by dexamethasone was reversed by simultaneous addition of PDGFBB. Suppressed proteoglycan production may affect the viscoelastic properties of tendon and increase the risk of spontaneous rupture.   ▪

Durable Mesenchymal Stem Cell Labelling by Using Polyhedral Superparamagnetic Iron Oxide Nanoparticles

Small polyhedral superparamagnetic iron oxide (SPIO) nanoparticles (<10 nm) coated with a thin layer of silica were prepared (SPIO@SiO(2) and SPIO@SiO(2)-NH(2)). Surface modification of the small polyhedral silica-coated SPIO nanoparticles with amines led to substantially higher mesenchymal stem cell (MSC) labelling efficiency without the use of additional transfecting agents. Therefore, amine surface-modified nanoparticles (SPIO@ SiO(2)-NH(2)) appeared to be the preferred candidate for MSC labelling. In vitro studies demonstrated that controlled labelling of SPIO@SiO(2) and SPIO@SiO(2)-NH(2) did not cause MSC death or proliferation inhibition. MSCs labelled with SPIO@SiO(2)-NH(2) nanoparticles retained differentiation potential and showed osteogenic, adipogenic and chondrogenic differentiations. The noncytotoxic polyhedral SPIO@SiO(2)-NH(2) nanoparticle-labelled MSCs were successfully implanted in rabbit brain and erector spinae muscle, and demonstrated long-lasting, durable MRI labelling efficacy after 8-12 weeks.

Abnormal melatonin receptor 1B expression in osteoblasts from girls with adolescent idiopathic scoliosis.

Abstract:  Melatonin signaling dysfunction has been associated with the etiology of adolescent idiopathic scoliosis (AIS). Genetic analysis has also associated the occurrence of AIS with the MT2 gene. Thus, we determined whether there is abnormality in the protein expression of melatonin receptors (MT) in AIS osteoblasts. In this study, we recruited 11 girls with severe AIS and eight normal subjects for intraoperative bone biopsies. MT1 and MT2 receptor protein expressions in the isolated osteoblasts were detected. Also, cell proliferation assay using different melatonin concentrations (0, 10−9, 10−5, 10−4 m) was carried out. The results showed that both MT1 and MT2 receptors are expressed in osteoblasts of the controls. While MT1 receptors were expressed in osteoblasts of all AIS subjects, osteoblasts of only 7 of 11 AIS showed expression of MT2 receptors. Melatonin stimulated control osteoblasts to proliferate. However, proliferation of AIS osteoblasts without expression of MT2 receptor, after treatment with melatonin, was minimal when compared with control and AIS osteoblasts with MT2 receptor expression. The proliferation of AIS osteoblasts with MT2 receptor was greater than those without. This is the first report demonstrating a difference between AIS and normal osteoblasts in the protein expression of MT2 receptor. The results suggest that there is a possible functional effect of MT2 receptor on osteoblast proliferation. AIS osteoblasts without expression of MT2 receptor showed the lowest percentage of viable cells after melatonin treatment. This possibly indicates the modulating role of melatonin through MT2 receptor on the proliferation of osteoblasts.

Abnormal proliferation and differentiation of osteoblasts from girls with adolescent idiopathic scoliosis to melatonin

Abstract:  Melatonin deficiency has been postulated as an etiologic factors in adolescent idiopathic scoliosis (AIS). In previous studies, melatonin was shown to regulate skeletal growth and bone formation in both humans and rats. Although it remains controversial whether there are differences in serum melatonin level between AIS and control subjects, melatonin signaling pathway dysfunction in osteoblasts has been reported in patients with AIS. Recently, our group found that melatonin receptor 1B (MT2) gene polymorphism was associated with the occurrence of AIS. Hence, the present study investigated the effect of melatonin on AIS osteoblasts. In vitro assays were performed with osteoblasts isolated from 17 severe AIS girls and nine control subjects. The osteoblasts were exposed to different concentrations of melatonin for 3 days. The effects of melatonin on cell proliferation (as evidenced by MTT assay) and differentiation (demonstrated by alkaline phosphatase activity) were determined. In the control group, melatonin significantly stimulated osteoblasts to proliferate and differentiate. However, in the AIS group, the stimulatory effects of melatonin were not discernible. Importantly, this finding demonstrated that there is a significant difference between AIS and control osteoblasts in functional response toward melatonin. Melatonin‐stimulated proliferation of control osteoblasts was inhibited by the MT2 antagonist, 4‐phenyl‐2‐propionamidotetraline, as well as by luzindole, a nonselective melatonin receptor antagonist, suggesting that MT2 is associated with the proliferative action of melatonin. The lack of response in AIS osteoblasts might be because of dysfunction of the melatonin signaling pathway, which may contribute to the low bone mineral density and abnormal skeletal growth observed in patients with AIS.

Correlation of Risser sign, radiographs of hand and wrist with the histological grade of iliac crest apophysis in girls with adolescent idiopathic scoliosis.

Study Design. A correlation analysis between histologic grade (HG) of iliac crest apophysis and skeletal age assessments. Objective. To study the correlation between HGs of iliac crest apophysis and the skeletal age of hand and wrist, digital skeletal age (DSA), and Risser sign in the girls with adolescent idiopathic scoliosis (AIS). Summary of Background Data. The evaluation of the remaining growth of the patients clinically and radiologically is highly significant in predicting the curve progression and thus influencing the treatment strategy in AIS. Methods. Fifty-three AIS girls with a mean age of 14.0 years undergoing corrective posterior spinal surgery and instrumentation with autogenous bone graft were recruited. Menarche status was recorded while the skeletal age of hand and wrist, DSA, and Risser grade were evaluated radiologically. Using a standardized HG of proliferative chondrocyte zone of the iliac crest apophysis, correlation between the HGs and the radiologic and clinical skeletal maturity parameters was analyzed. Results. The HGs were negatively correlated with the radiologic parameters with the highest correlation coefficient between HGs and skeletal age of hand and wrist. The negative correlation between HGs and the 2 clinical parameters was significant and could be enhanced by combining with the radiologic parameters. No proliferative chondrocyte zone of the apophysis was detected when patients were either over 16 years of skeletal age or Risser grade 5, as well as 2-year postmenarche or over 15 years of chronological age in patients with DSA stage III and Risser grade 4. Conclusion. Radiologic skeletal age of the wrist and digits can provide important information for maturity assessment in girls with AIS. It can also enhance the sensitivity of clinical parameters in determining the remaining growth potential.

Bone densitometry: which skeletal sites are best predicted by bone mass determinants?

Evaluation of bone mineral content/bone mass density (BMC/BMD) is important to determine bone mass development among adolescents in health and disease. It is uncertain at which skeletal site BMC/BMD is best predicted by bone mass determinants. On the other hand, intrapersonal BMC/BMD data can be clustered into a composite index score to facilitate correlation and outcome prediction analysis. This study aimed to identify the skeletal site that was best predicted by bone mass determinants and to develop a composite index score based on multisite BMC/BMD values in healthy adolescent girls. Eleven BMD/BMC variables per subject were evaluated by using dual-energy X-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT) in 236 healthy girls aged 12–15 years. Bone mass determinants, namely, weight, height, puberty, dietary calcium, physical activity, and bone turnover markers, were determined. Factor analysis was used to develop composite index scores that summarized characteristics of multisite BMC/BMD. Results showed that lumbar spinal BMD and BMC (by DXA) and tibial integral BMD (by pQCT) were the BMC/BMD sites better predicted by bone mass determinants (R2, 0.57–0.77) in multiple regression analysis. On the other hand, three composite index scores representing areal BMD, areal BMC, and vBMD were derived to summarize the original BMC/BMD values. The composite index scores had similar predicting power (R2, 0.419–0.749) compared to those of original BMC/BMD, indicating that the composite index scores were representative of the original variables. To conclude, lumbar spinal BMD and BMC and tibial integral BMD were the three BMC/BMD variables better predicted by bone mass determinants. This evaluation would help select appropriate skeletal sites as outcome measures for bone mass evaluation in future studies. Also, the development of composite index scores could help reduce the number of variables for correlation and outcome prediction analyses.

Are volumetric bone mineral density and bone micro-architecture associated with leptin and soluble leptin receptor levels in adolescent idiopathic scoliosis?--A case-control study.

Background Adolescent idiopathic scoliosis (AIS) is associated with low bone mineral density (BMD). The underlying etiology and how it may relate to the development of osteopenia remains unknown. Leptin has been postulated as one of the etiologic factors of AIS because of its profound effects on bone metabolism and pubertal growth. Its modulator, soluble leptin receptor (sOB-R), may affect leptin bioavailability and signaling. This study aimed to investigate whether serum leptin and sOB-R levels may be associated with bone quality, and whether these relationships may differ between young adolescent girls with and without AIS. Methods This was a case-control study involving 94 newly diagnosed AIS girls (Cobb angle 12–48°) aged 12 to 14 years old and 87 age and gender-matched normal controls. Subjects with BMI>23.0 Kg/m2 were excluded. Anthropometric measurements including body weight, height, arm span and sitting height were taken. Serum total leptin and sOB-R were assayed with ELISA. Non-dominant distal radius was scanned with High Resolution pQCT for assessing bone quality in terms of bone morphometry, volumetric BMD (vBMD) and trabecular bone micro-architecture. Results Compared with normal controls, AIS girls had numerically higher sOB-R (p = 0.006), lower average vBMD (p = 0.048), lower cortical vBMD (p = 0.029), higher cortical bone perimeter (p = 0.014) and higher trabecular area (p = 0.027), but none remained statistically significant after the Hochberg-Benjamini procedure. Correlation analysis on serum leptin level indicated that distinctive correlations with trabecular bone parameters occurred only in AIS. Conclusion This study showed that bone quality in AIS girls was deranged as compared with controls. In addition, the distinct differences in correlation pattern between leptin and trabecular bone parameters indicated possible abnormalities in bone metabolism and dysfunction of the leptin signaling pathway in AIS.

Effects of oxygen-induced lung damage on megakaryocytopoiesis and platelet homeostasis in a rat model

The association between lung injury and thrombocytopenia was investigated by comparing the megakaryocyte and platelet counts, and platelet activation using P-selectin as a marker, between the prepulmonary (right atrial) and postpulmonary (left atrial) blood in adult and neonatal (preterm and term) rats with and without hyperoxic lung injury. In the healthy controls, the postpulmonary blood had lower megakaryocyte count (prepulmonary versus postpulmonary: Preterm: 8.7[0.6]versus 3.9[0.3] per ml, p < 0.001; Term: 8.7[1.1]versus 2.6[0.4] per ml, p < 0.001; Adult: median [interquartile ranges]: 2.5[1.0, 5.0]versus 1.0[0, 3.0] per ml, p < 0.001), higher platelet count (prepulmonary versus postpulmonary: Preterm: 491.2[11.1] × 109/L versus 595.1[10.2] × 109/L, p < 0.001; Term: 472.5[19.9] × 109/L versus 579.3[26.2] × 109/L, p < 0.001; Adult: 513.9[31.5] × 109/L versus 664.7[28.8] × 109/L, p < 0.001), but similar P-selectin expression. In contrast, the lung-damaged animals did not show any such differences in either megakaryocyte or platelet count, but P-selectin expression was greater in the postpulmonary blood (prepulmonary versus postpulmonary: Preterm: 38.7[3.9]versus 56.4[4.9;[percnt; platelets, p = 0.02; Term: 40.9[2.0]versus 54.0[4.2;[percnt; platelets, p = 0.002; Adults: 30.0[3.6]versus 49.1[4.7;[percnt; platelets, p = 0.003). Peripheral platelet and intra-pulmonary megakaryocyte counts in the lung-damaged rats were significantly lower than those in their respective controls. Intra-pulmonary thrombi or platelet aggregation were detected in the lung-damaged rats but not in the controls. These findings showed that hyperoxic lung damage reduced circulating platelets through (1) failure of the lungs to retain and fragment megakaryocytes to release platelets, and (2) platelet activation leading to platelet aggregation, thrombi formation and platelet consumption. The magnitude of platelet reduction was physiologically significant, as demonstrated by higher counts of megakaryocyte colony forming units in the bone marrow culture of the animals in the hyperoxia group when compared with the controls.

Lower Muscle Mass and Body Fat in Adolescent Idiopathic Scoliosis Are Associated With Abnormal Leptin Bioavailability.

Study Design. This was a case-control study. Objective. This study aimed to investigate the body composition and its correlation with leptin and soluble leptin receptor (sOB-R) levels in girls with adolescent idiopathic scoliosis (AIS) and compared with healthy controls. Summary of Background Data. Patients with AIS are associated with lower body weight, taller stature, lower body mass index (BMI), and deranged bone quality. Despite the widely reported lower BMI and body weight in girls with AIS, the body composition of these patients was not thoroughly studied with sufficient sample size. Leptin is an important factor in regulating energy and bone metabolism, and has been postulated as one of the etiologic factors of AIS. Methods. One hundred forty-eight AIS and 116 control girls aged 12 to 14 were recruited. Body composition was measured with bioelectrical impedance analysis. Caloric intake and physical activity level were assessed by food frequency and Baecke questionnaires respectively. Serum total leptin and sOB-R levels were measured with enzyme-linked immunosorbent assay, and free leptin index was calculated. Results. AIS girls had lower body weight and BMI, other anthropometric and sexual maturity parameters were comparable with controls. There were no difference in caloric intake and physical activity levels. After adjustment for physical activity level, AIS girls had lower skeletal muscle mass, lower body fat, and %body fat. Higher sOB-R and lower free leptin index were found in AIS girls after adjusted for age and body weight. Weaker correlations between serum total leptin, FLI, and body composition parameters were observed in AIS girls. Conclusion. Results suggested that the lower body weight in AIS girls was contributed by both lower skeletal muscle mass and lower body fat. Altered leptin bioavailability also exists in AIS girls and could lead to lower body weight, lower BMI, and abnormal body composition that were manifested in AIS simultaneously. Level of Evidence: 4