The influence of friedelin, resinone, tingenone and betulin of compounds on chondrogenic differentiation of porcine adipose-derived mesenchymal stem cells (pADMSCs).

The study investigated the influence of friedelin, resinone, tingenone and betulin plant-based secondary metabolite compounds on cellular proliferation, extracellular matrix (ECM) components synthesis, expression of chondrogenic markers and maturation of differentiated chondrocytes (cell proliferation and hypertrophy) in porcine adipose-derived mesenchymal stem cells (pADMSCs) undergoing chondrogenic differentiation. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Cyquant assays were used to determine cell proliferation, viability, and total cellular DNA, DMMB (Dimethyl methylene blue) was used for glycosaminoglycan (GAG) synthesis, RT-qPCR for gene expression and histology combined with immunohistochemistry for cartilage ECM proteoglycan deposition. The MTT results showed that friedelin at 37 µM, resinone at 36 µM and betulin at 18 µM with cell viability of above 100% compared to control. Tingenone at 37 µM showed cell viability of about 76%. These concentrations were considered the most effective with no toxicity effect on the cells and were further analysed with TGF-ß3 (10 ng/mL) as a positive control. The results showed a high synthesis of DNA with friedelin on day 14. There was up-regulation of SOX 9, Col II and Col X with friedelin and resinone at day 14 with the significance of p < 0.01. Pellet from friedelin, resinone and tingenone showed more staining of the matrix for Safranin-O and Toluidine blue at day 14. Immunohistostaining of collagen type X (COL-10) showed more stain intensity at friedelin and resinone on day 21. These results provided new knowledge on the potential use of natural isolated secondary metabolites compounds as inducers for chondrogenic and bone differentiation.

Osteoinductive Activity of Selected Demineralized Bone Matrix Products from Donors of Different Ages.

BACKGROUND: Despite previous confirmation of osteoinductive potential of demineralized bone matrix (DBM) by other researchers, there is not yet any evidence of studies showing the osteoinductive activity of DBM products from South Africa tissue banks using both in vitro and animal models. This work evaluated the osteoinductivity of DBM both in vitro and in vivo. METHOD: DBM particles from six donors from the Centre for Tissue Engineering and C2C12 were cultured (5x104) in 24-well plates using DMEM/F-12 medium supplemented with 10% FBS. After 24 h medium was replaced with medium containing 1% FBS and 5 mg/ml of DBM particles. Bone morphogenetic protein-2 (BMP-2,500 pg/ml) was used as a positive control. After 48 h of incubation, cells were assayed for osteoinductive potentials. In an in vivo study, 27 Wistar rats aged six to eight weeks were divided into three groups and experimentally observed for 7, 14 and 28 days. Implants were explanted according to the duration of the experiment. RESULTS: Increase in cell growth was observed in C2C12 treated with DBM samples. BMP-2 and DBM samples were found to stimulate alkaline phosphatase activity and ELISA assay. Animal weight increase was observed during the 7, 14 and 28 days. Cartilage regeneration were also observed in the histology results. CONCLUSION: BMP-2 played a role in the differentiation of myoblast cells into osteoblasts. DBM products showed different osteoinductive capacity both in vitro and in vivo. Findings were variable and time-dependent. From our results, this study supports the effectiveness of DBM fromdonors aged between 45 to 55 years.

Effects of resveratrol on collagen type II protein in the superficial and middle zone chondrocytes of porcine articular cartilage.

ETHNOPHARMACOLOGICAL RELEVANCE: Resveratrol (RSV) was first isolated in 1940 from the roots of white hellebore (Veratrum grandiflorum (Maxim. ex Miq) O. Loes) and in 1963 from the roots of Japanese knotweed (Polygonum cuspidatum Siebold & Zucc.). These species have been used traditionally to treat arthritis, gout or inflammation. RSV (3,5,4-trihydroxystilbene) is a polyphenolic phytoalexin compound found in various plants, such as grape vines, berries, peanuts, seeds and roots; the highest concentration is in the skin of red grapes. This component of red wine has potent anti-inflammatory properties and may reduce the side effects of non-steroidal anti-inflammatory drugs that are currently used for pain amelioration in osteoarthritis (OA). In early degeneration of articular cartilage, which may lead to OA there is a loss of the tensile properties, indicative of damage to the fibrillar network. Damage to this fibrillar meshwork, made up of primarily collagen type II (90-95%), may be a critical event in the pathology of many arthritides, due in part to the very slow rate of collagen turnover within the cartilage. Collagen type II is the pre-dominant protein of the cartilage middle zone matrix mainly responsible for tensile strength of articular cartilage. The aim of the study was to investigate the effects of RSV on the expression of collagen type II from the superficial and middle zone chondrocytes of porcine articular cartilage. MATERIALS AND METHODS: Porcine articular chondrocytes were isolated from the superficial and middle zone of articular cartilage, cultured as monolayers in serum-free chemically defined medium for four days. Effects of RSV on porcine articular chondrocytes were studied by assessing expression of collagen type II mRNA by RT-PCR and protein levels of collagen type II by ELISA; as well as localisation of collagen type II on cartilage tissue sections using immunohistochemistry. RESULTS: RSV significantly stimulated the expression of collagen type II at the mRNA and protein levels in the superficial and middle zone. Immunohistochemistry revealed that collagen type II was present along the whole cartilage tissue sections. The staining was strong in the superficial zone, mild in the middle zone and less around hypertrophic chondrocytes in the deep zone. Histological analysis confirmed that cartilage slices were obtained from specific articular cartilage zones. CONCLUSION: This study revealed the importance of RSV in the regulation of collagen type II protein in different zones of articular cartilage.

Antimicrobial and anti-inflammatory activities of Pleurostylia capensis Turcz (Loes) (celastraceae).

BACKGROUND: Pleurostylia capensis is a large tree that can reach the maximum height of 20 m long, and it have been traditionally used as cosmetic, for steam bath, ritual body wash, and as a purgative to treat symptoms of witchcraft. Using ethanol, chloroform, dichloromethane (DCM), ethyl acetate (EA), and water extracts, leaves, bark and roots of Pleurostylia capensis were investigated scientifically for their effectiveness in antimicrobial, antioxidant and anti-inflammatory activities using standard methods. MATERIALS AND METHODS: The extracts were evaluated for antimicrobial activity against Gram positive (Staphylococcus aureus, Bacillus cereus, and Mycobacterium smegmatis), Gram negative (Escherichia coli, Klebsiella pneumonia, Klebsiella oxytoca, Streptococcus pyogenes, Pseudomonas aeruginosa and Salmonella typhimurium), and Candida albicans. The antioxidant activity was investigated using 2, 2-diphenlyl-1-picrylhadrazyl (DPPH), free radical scavenging assay. The anti-inflammatory activity of P. capensis extracts was evaluated against both cyclooxygenase enzymes (COX 1 and 2). RESULTS: The ethyl acetate extracts of P. capensis showed a strong antimicrobial activity against B. cereus, K. pneumonia, S. pyogenes, and M. smegmatis with MIC value of 0.39 and 0.78 mg/ml. While the ethanol bark extract was most active against M. smegmatis with MIC value of 0.78 mg/ml; the least potent activity was observed with dichloromethane, chloroform and water extracts, with an MIC value ranging from 1.56 mg/ml to 50.0 mg/ml. The plant extracts proved to be good antioxidant agent, whereas extracts of ethanol were the most active, with IC50 ranging from 1.00 to 1.74 µg/ml, which is lower, and in close range to Vitamin C (1.40 µg/ml). CONCLUSIONS: Its moderation to potent inhibitory activity was observed in all extracts. Ethanol and dichloromethane extracts were among the most potent when compared to water and petroleum ether extracts. The water extracts showed to be nontoxic on the Hek cell line with an IC50 value of 204.0, and 207.3 µg/ml (roots and bark) respectively. The dichloromethane, ethyl acetate, chloroform and ethanol extracts showed to be toxic on the Hek cell, with IC50 range from 5.94 to 42.91µg/ml. The results obtained indicate the effectiveness of these plants.