Materials and Methods: Thirty-six female Sprague-Dawley rats were divided into six groups: Sham-operated (SHAM), OVX control, OVX and given Premarin at 64.5 µg/kg (OVX+E2), OVX and given VCO at 4.29 ml/kg (OVX+V), OVX and given TRF at 30 mg/kg (OVX+T), and OVX and given a combination of VCO at 4.29 ml/kg and TRF at 30 mg/kg (OVX+VT). Following 24 weeks of treatments, blood and femora samples were taken for analyses.
Results: There were no significant differences in serum osteocalcin levels between the groups (p>0.05), while serum C-terminal telopeptide of Type I collagen levels of the OVX+VT group were significantly lower than the other groups (p<0.05). The dynamic bone histomorphometry analysis of the femur showed that the double-labeled surface/bone surface (dLS/BS), mineral apposition rate, and bone formation rate/BS of the OVX+E2, OVX+T, and OVX+VT groups were significantly higher than the rest of the groups (p<0.05).
Conclusion: A combination of VCO and TRF has the potential as a therapeutic agent to restore bone loss induced by ovariectomy and high-fat diet.
Methods: The genes were transferred into chondrocytes at passage-1 (P1) via lipofection. The post-transfected chondrocytes (SOX9-, TERT- and SOX9/TERT) were analysed at P1, P2 and P3. The non-transfected group was used as control. The 3D culture was established using the chondrocytes seeded in a disc-shaped PLGA/fibrin and PLGA scaffolds. The resulting 3D "cells-scaffolds" constructs were analysed at week-1, -2 and -3. The histoarchitecture was evaluated using haematoxylin and eosin, alcian blue and safranin o stains. The quantitative sulphated glycosaminoglycan (sGAG) content was measured using biochemical assay. The cartilage-specific markers expression were analysed via real-time polymerase chain reaction.
Results: All monolayer cultured chondrocytes showed flattened, fibroblast-like appearance throughout passages. Proteoglycan and sGAG were not detected at the pericellular matrix region of the chondrocytes. The sGAG content assay indicated the matrix production depletion in the culture. The cartilage-specific markers, COL2A1 and ACAN, were downregulated. However, the dedifferentiation marker, COL1A1 was upregulated. In 3D "cells-scaffolds" constructs, regardless of transfection groups, chondrocytes seeded in PLGA/fibrin showed a more uniform distribution and produced denser matrix than the PLGA group especially at week-3. Both sGAG and proteoglycan were clearly visualised in the constructs, supported by the increment of sGAG content, quantitatively. Both COL2A1 and ACAN were upregulated in SOX9/TERT-PLGA and SOX9/TERT-PLGA/fibrin respectively. While, COL1A1 was downregulated in SOX9/TERT-PLGA.
Conclusion: These findings indicated that the SOX9/TERT-transfected chondrocytes incorporation into 3D scaffolds facilitates the cartilage regeneration which is viable structurally and functionally.
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.
METHODS: This study was conducted between the years 2014 to 2016 at the Tissue Engineering Centre, UKM Medical Centre. OTC-I was extracted from ovine tendon, and fabricated into 3D scaffolds in the form of sponge, hydrogel and film. A polystyrene surface coated with OTC-I was used as the 2D culture condition. Genipin was used to crosslink the OTC-I. A non-coated polystyrene surface was used as a control. The mechanical strength of OTC-I scaffolds was evaluated. Attachment, proliferation and morphological features of HDF were assessed and compared between conditions.
RESULTS: The mechanical strength of OTC-I sponge was significantly higher than that of the other scaffolds. OTC-I scaffolds and the coated surface significantly enhanced HDF attachment and proliferation compared to the control, but no differences were observed between the scaffolds and coated surface. In contrast, the morphological features of HDF including spreading, filopodia, lamellipodia and actin cytoskeletal formation differed between conditions.
CONCLUSION: OTC-I can be moulded into various scaffolds that are biocompatible and thus could be suitable as scaffolds for developing tissue substitutes for clinical applications and in vitro tissue models. However, further study is required to determine the effect of morphological properties on the functional and molecular properties of HDF.
MATERIALS AND METHODS: Chondrocytes were isolated from the costal cartilage of newborn rats using 0.15% collagenase solution in DMEM. The cells was characterized by glycosaminoglycan staining with alcian blue. Chondrocyte scaffolds were obtained from 4% type I porcine atelocollagen and 10% GelMA by micromolding and then implanted subcutaneously into the withers of two groups of Wistar rats. Histological and immunohistochemical studies were performed on days 12 and 26 after implantation. Tissue samples were stained with hematoxylin and eosin, alcian blue; type I and type II collagens were identified by the corresponding antibodies.
RESULTS: The implanted scaffolds induced a moderate inflammatory response in both groups when implanted in animals. By day 26 after implantation, both collagen and GelMA had almost completely resorbed. Cartilage tissue formation was observed in both animal groups. The newly formed tissue was stained intensively with alcian blue, and the cells were positive for both types of collagen. Cartilage tissue was formed among muscle fibers.
CONCLUSION: The ability of collagen type I and GelMA hydrogels to form hyaline cartilage in animals after subcutaneous implantation of scaffolds was studied. Both collagen and GelMA contributed to formation of hyaline-like cartilage tissue type in animals, but the chondrocyte phenotype is characterized as mixed. Additional detailed studies of possible mechanisms of chondrogenesis under the influence of each of the hydrogels are needed.
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: Lizardfish bone collagens were extracted with various acids (i.e., acetic, lactic and citric acids). All extraction processes were conducted in a chiller room (4 °C). The extracted collagens were biochemically characterized, such as hydroxyproline content, Ultraviolet (UV) absorption, X-ray diffraction (XRD), Fourier transform infrared spectroscopy spectra (FTIR), Differential scanning calorimetry (DSC) and solubility in different pH values and NaCl concentrations.
RESULTS: The yield of extracted collagens ranged between 1.73% and 2.59%, with the highest (p collagen (CaEC). Protein patterns confirmed that all-collagen samples had two identical subunits, α1 and α2, representing type I collagen. The highest whiteness value was found in acetic acid-extracted collagen (AaEC), but there was no significant difference (p ≥ 0.05) compared to lactic acid-extracted collagen (LaEC). UV absorption and XRD analysis reflected the characteristics of the collagen, as reported in the literature. For the FTIR, all acid-extracted collagen samples presented a triple helical structure. The thermal transition temperature (T max = 77.92-89.04 °C) was in accordance with collagen extracted from other fish species. All extracted collagens were highly soluble in acidic pH and low concentrations of NaCl (0-20 g/L). In conclusion, collagens extracted from lizardfish bone may be used as alternative sources of collagen in industrial settings, and AaEC would be considered superior in terms of the characteristics evaluated in this study.
METHODS: Fifty-six female Sprague-Dawley rats were randomly allocated into eight groups (n = 7): SHAM (healthy sham control); OVX (ovarietomized) nontreated rats (negative control); OVX + Remifemin (100 mg/kg body weight), and 2% green tea extract (positive controls); OVX + OS 50% ethanolic and aqueous extracts, both at either 150 or 300 mg/kg. After 16 weeks, the rats' bones and blood were evaluated for osteoporosis indicators (protein and mRNA expressions), micro-computed tomography for bone histomorphometry, and three-point bending test for tibia mechanical strength.
RESULTS: The extracts dose-dependently and significantly (P collagen-1 synthesis (collagen type 1 alpha-1) mRNA expressions, and down-regulated bone resorption (TNFSF11 and nuclear factor-kappa B) mRNA expressions. Both the water and 50% ethanolic extract were effective. The effective dose is equivalent to 25 to 50 mg/kg extract for humans.
CONCLUSIONS: The extract showed bone-protective and antiosteoporotic effects (improving bone strength, flexibility, bone density, and bone morphometry) by reducing inflammation and the bone resorption biomarkers, while enhancing bone formation biomarkers and collagen synthesis.