MATERIALS AND METHODS: Murine MC3T3-E1 preosteoblastic cells were cultured in the different concentrations of AnTT (0.001-1 µg/mL) up to 24 days. Expression of osteoblastic differentiation markers was measured by qPCR (osterix [OSX], collagen 1 alpha 1 [COL1α1], alkaline phosphatase [ALP], and osteocalcin [OCN]) and by fluorometric assay for ALP activity. Detection of collagen and mineralized nodules was done via Direct Red staining and Alizarin Red staining, respectively.
RESULTS: The results showed that osteoblastic differentiation-related genes, such as OSX, COL1α1, ALP, and OCN, were significantly increased in the AnTT-treated groups compared to the vehicle group in a time-dependent manner (P<0.05). Type 1 collagen level was increased from day 3 to day 15 in the AnTT-treated groups, while ALP activity was increased from day 9 to day 21 in the AnTT-treated groups (P<0.05). Enhanced mineralization was observed in the AnTT-treated groups via increasing Alizarin Red staining from day 3 to day 21 (P<0.05).
CONCLUSION: Our results suggest that AnTT enhances the osteogenic activity by promoting the bone formation-related genes and proteins in a temporal and sequential manner.
OBJECTIVE(S): The present study was aimed to investigate the mechanism of bone-forming capacity of EL using MC3T3-E1 as an in vitro osteoblastic model.
MATERIALS AND METHODS: The cell differentiation capacity of EL was investigated by evaluating cell growth, alkaline phosphatase (ALP) activity, collagen deposition and mineralization. Taken together, time-mannered expression of bone-related mediators which include bone morphogenic protein-2 (BMP-2), ALP, runt-related transcription factor-2 (Runx-2), osteocalcin (OCN), type I collagen, osteopontin (OPN), transforming growth factor-β1 (TGF-β1) and androgen receptor (AR) were measured to comprehend bone-forming mechanism of EL.
RESULTS: Results demonstrated a superior cell differentiation efficacy of EL (particularly at a dose of 25 μg/mL) that was evidenced by dramatically increased cell growth, higher ALP activity, collagen deposition and mineralization compared to the testosterone. Results analysis of the bone-related protein biomarkers indicated that the expression of these mediators was well-regulated in EL-treated cell cultures compared to the control groups. These findings revealed potential molecular mechanism of EL for the prevention and treatment of male osteoporosis.
CONCLUSION: The resulting data suggested that EL exhibited superior efficacy in stimulating bone formation via up-regulating the expression of various mitogenic proteins and thus can be considered as a potential natural alternative therapy for the treatment of osteoporosis.
METHODS: One hundred and ninety-seven healthy women, aged 25 to 60, were selected from a hospital staff health screening program; 68% were Chinese, 18% Malay, and 14% Indian. P1NP, CTX, and 25-OHD(3) were measured using the Roche Cobas® electrochemiluminescence immunoassay. Serum PTH was measured using the Siemens ADVIA Centaur® immunoassay.
RESULTS: Sixty-five percent had 25-OHD(3) concentrations <50 nmol/l. Vitamin D insufficiency (25-OHD(3) < 50 nmol/l) was more prevalent in Malays (89%) and Indians (82%) compared to Chinese (56%). There was no correlation between vitamin D and age. PTH positively correlated with age, and Malays and Indians had higher PTH concentrations than Chinese. There was an inverse correlation between PTH and 25-OHD(3), but no threshold of 25-OHD(3) concentrations at which PTH plateaued. The bone turnover markers P1NP and CTX inversely correlated with age but were not different between ethnic groups. CTX and P1NP exhibited good correlation, however, there was no significant correlation between 25-OHD(3) or PTH concentrations and the bone turnover markers P1NP and CTX.
CONCLUSIONS: Healthy women in Singapore have a high prevalence of vitamin D insufficiency. Vitamin D insufficiency was more prevalent in Malays and Indians compared to Chinese.
Methods: hTenowere isolated from human hamstring tendon. Presence of insulin receptor beta (INSR-β) on normal tendon tissues and the hTeno monolayer culture were analyzed by immunofluorescence staining. The presence of Glucose Transporter Type 1 (GLUT1) and Glucose Transporter Type 4 (GLUT4) on the hTeno monolayer culture were also analyzed by immunofluorescence staining. Primary hTeno were treated with 0.008, 0.08, 0.8 and 8.0 µM of TNF-α, with and without insulin supplement. Outcome measures include 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) assay to determine the glucose uptake activity; colourimetric total collagen assay to quantify the total collagen expression levels; COL-I ELISA assay to measure the COL-I expression levels and real-time qPCR to analyze the mRNA gene expressions levels of Scleraxis (SCX), Mohawk (MKX), type I collagen (COL1A1), type III collagen (COL3A1), matrix metalloproteinases (MMP)-9 and MMP-13 in hTeno when treated with TNF-α. Apoptosis assay for hTeno induced with TNF-α was conducted using Annexin-V FITC flow cytometry analysis.
Results: Immunofluorescence imaging showed the presence of INSR-β on the hTeno in the human Achilles tendon tissues and in the hTeno in monolayer culture. GLUT1 and GLUT4 were both positively expressed in the hTeno. TNF-α significantly reduced the insulin-mediated 2-NBDG uptake in all the tested concentrations, especially at 0.008 µM. Total collagen expression levels and COL-I expression levels in hTeno were also significantly reduced in hTeno treated with 0.008 µM of TNF-α. The SCX, MKX and COL1A1 mRNA expression levels were significantly downregulated in all TNF-α treated hTeno, whereas the COL3A1, MMP-9 and MMP-13 were significantly upregulated in the TNF-α treated cells. TNF-α progressively increased the apoptotic cells at 48 and 72 h.
Conclusion: At 0.008 µM of TNF-α, an IR condition was induced in hTeno, supported with the significant reduction in glucose uptake, as well as significantly reduced total collagen, specifically COL-I expression levels, downregulation of candidate tenogenic markers genes (SCX and MKX), and upregulation of ECM catabolic genes (MMP-9 and MMP-13). Development of novel IR model in hTeno provides an insight on how tendon homeostasis could be affected and can be used as a tool for further discovering the effects on downstream molecular pathways, as the implication for diabetic tendinopathy.
METHODS: The effective dose of Ecklonia cava phlorotannins (ECP) for hyperglycaemic wound healing was determined prior to phlorotannin nanofibre fabrication using polyvinyl-alcohol (PVA), polyvinylpyrrolidone (PVP), and ECP. Vapour glutaraldehyde was used for crosslinking of the PVA/PVP nanofibres. The phlorotannin nanofibres were characterised, and their safety and cytocompatibility were validated. Next, the wound healing effect of phlorotannin nanofibres was determined with 2D wound scratch assay, whereas immunofluorescence staining of Collagen-I (Col-I) and Cytokeratin-14 (CK-14) was performed in human dermal fibroblasts (HDF) and human epidermal keratinocytes (HEK), respectively.
RESULTS: Our results demonstrated that 0.01 μg/mL ECP significantly improved hyperglycaemic wound healing without compromising cell viability and proliferation. Among all nanofibres, PVA/PVP/0.01 wt% ECP nanofibres exhibited the best hyperglycaemic wound healing effect. They displayed a diameter of 334.7 ± 10.1 nm, a porosity of 40.7 ± 3.3%, and a WVTR of 1718.1 ± 32.3 g/m2/day. Besides, the FTIR spectra and phlorotannin release profile validated the successful vapour glutaraldehyde crosslinking and ECP incorporation. We also demonstrated the potential of phlorotannin nanofibres as a non-cytotoxic wound dressing as they support the viability and proliferation of both HDF and HEK. Furthermore, phlorotannin nanofibres significantly ameliorated the impaired hyperglycaemic wound healing and restored the hyperglycaemic-induced Col-I reduction in HDF.
CONCLUSION: Taken together, our findings show that phlorotannin nanofibres have the potential to be used as a diabetic wound dressing.