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Fulltext Stichopus chloronotus aqueous extract as a chondroprotective agent for human chondrocytes isolated from osteoarthitis articular cartilage in vitro

Mohd Heikal MY, Ahmad Nazrun S, Chua KH, Norzana AG

Cytotechnology, 2019 Apr;71(2):521-537.
PMID: 30719603 DOI: 10.1007/s10616-019-00298-2

The proinflammatory cytokines, metalloproteinases family (MMPs), inflammatory mediators PGE2, COX-2 and NO are the most important group of compounds responsible for the loss of metabolic homeostasis of articular cartilage by promoting catabolic and destructive processes in the pathogenesis of osteoarthritis (OA). Stichopus chloronotus, a marine sea cucumber which is rich in n-3 PUFAs and phenolic compound, may exert a favorable influence on the course of the disease. The objective of this study was to investigate the regeneration and anti-inflammatory potential of S. chloronotus aqueous extract (SCAE) on human OA articular chondrocytes (HOC).
METHODS: The HOC isolated from knee joint cartilage removed during surgery were cultured with SCAE for 7 days. The effect of SCAE on anabolic and catabolic gene expression was verified by real-time PCR. Monolayer chondrocytes were stained with toluidine blue whereas sGAG, NO and PGE2 production in medium were analyzed by ELISA.
RESULTS: The HOC cultured in various SCAE have polygonal morphology maintaining their chondrocytes characteristic. SAE supplementation tested was found to be effective pro-chondrogenic, anti-inflammatory and anti-oxidative agents, as evidenced by upregulation of cartilage specific markers collagen type II, aggrecan core protein and sox-9 expression and downregulation of collagen type 1, IL-1, IL-6, IL-8, MMP-1, MMP-3, MMP-13, COX-2, iNOS and PAR-2 expression. The presence of SCAE in the culture was able to increase sGAG and reduce NO and PGE2 production significantly.
CONCLUSIONS: These results suggested that SCAE demonstrated chondroprotective ability by suppressing catabolic activities, oxidative damage and effectively promoting chondrocytes growth.
Autologous implantation of bilayered tissue-engineered respiratory epithelium for tracheal mucosal regenesis in a sheep model

Mohd Heikal MY, Aminuddin BS, Jeevanan J, Chen HC, Sharifah SH, Ruszymah BH

Cells Tissues Organs (Print), 2010;192(5):292-302.
PMID: 20616535 DOI: 10.1159/000318675

The objective of this study was to regenerate the tracheal epithelium using autologous nasal respiratory epithelial cells in a sheep model. Respiratory epithelium and fibroblast cells were harvested from nasal turbinates and cultured for 1 week. After confluence, respiratory epithelium and fibroblast cells were suspended in autologous fibrin polymerized separately to form a tissue-engineered respiratory epithelial construct (TEREC). A 3 × 2 cm² tracheal mucosal defect was created, and implanted with TEREC and titanium mesh as a temporary scaffold. The control groups were divided into 2 groups: polymerized autologous fibrin devoid of cells (group 1), and no construct implanted (group 2). All sheep were euthanized at 4 weeks of implantation. Gross observation of the trachea showed minimal luminal stenosis formation in the experimental group compared to the control groups. Macroscopic evaluation revealed significant mucosal fibrosis in control group 1 (71.8%) as compared to the experimental group (7%). Hematoxylin and eosin staining revealed the presence of minimal overgrowth of fibrous connective tissue covered by respiratory epithelium. A positive red fluorescence staining of PKH26 on engineered tissue 4 weeks after implantation confirmed the presence of cultured nasal respiratory epithelial cells intercalated with native tracheal epithelial cells. Scanning electron microscopy showed the presence of short microvilli representing immature cilia on the surface of the epithelium. Our study showed that TEREC was a good replacement for a tracheal mucosal defect and was able to promote natural regenesis of the tracheal epithelium with minimal fibrosis. This study highlighted a new technique in the treatment of tracheal stenosis.
Comparison of the effects between animal-derived trypsin and recombinant trypsin on human skin cells proliferation, gene and protein expression

Manira M, Khairul Anuar K, Seet WT, Ahmad Irfan AW, Ng MH, Chua KH, et al.

Cell Tissue Bank, 2014 Mar;15(1):41-9.
PMID: 23456438 DOI: 10.1007/s10561-013-9368-y

Animal-derivative free reagents are preferred in skin cell culture for clinical applications. The aim of this study was to compare the performance and effects between animal-derived trypsin and recombinant trypsin for skin cells culture and expansion. Full thickness human skin was digested in 0.6 % collagenase for 6 h to liberate the fibroblasts, followed by treatment with either animal-derived trypsin; Trypsin EDTA (TE) or recombinant trypsin; TrypLE Select (TS) to liberate the keratinocytes. Both keratinocytes and fibroblasts were then culture-expanded until passage 2. Trypsinization for both cell types during culture-expansion was performed using either TE or TS. Total cells yield was determined using a haemocytometer. Expression of collagen type I, collagen type III (Col-III), cytokeratin 10, and cytokeratin 14 genes were quantified via RT-PCR and further confirmed with immunocytochemical staining. The results of our study showed that the total cell yield for both keratinocytes and fibroblasts treated with TE or TS were comparable. RT-PCR showed that expression of skin-specific genes except Col-III was higher in the TS treated group compared to that in the TE group. Expression of proteins specific to the two cell types were confirmed by immunocytochemical staining in both TE and TS groups. In conclusion, the performance of the recombinant trypsin is comparable with the well-established animal-derived trypsin for human skin cell culture expansion in terms of cell yield and expression of specific cellular markers.

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