Chicken type 2 Sternum Collagen Type II

Tissue engineering is a technique that can be used for repairing damaged cartilage. The aim of this study was chondro-differentiation of mesenchymal stem cells (MSCs) on polyvinyl alcohol/gelatin/chondroitin sulfate (PVA/GT/CS) blend nanofibers. Nanofibers were fabricated by electrospinning method with different concentrations of CS (10%, 15%, and 20%). MSCs were seeded on nanofibers for biocompatibility and cell viability test by MTT assay. Alcian blue staining assay was done to show that MSCs were differentiated to chondrocyte by staining the glycosaminoglycans (GAGs) that was produced by cells. Reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry (ICC) assay were done to confirm type II collagen expression. Scaffolds with different concentrations of CS were tested for biocompatibility, and the nanofiber with 15% of GAG showed better results (P = 0.02) compared with other nanofibers; therefore, it was chosen as the main sample for chondrogenesis tests. On the basis of scanning electron microscopy (SEM), hMSCs had a proper attachment to the nanofiber with 15% CS. Alcian blue staining showed the different rates of blue coloration, which means cells produced different types of GAGs from the GAG we used in the nanofiber. RT-PCR and ICC showed that COL2A1 was expressed in mRNA and protein levels. This study indicates that the PVA/GT/CS nanofiber containing 15% CS can be a good candidate for cartilage tissue engineering.

Purpose To identify the genetic defect causing early-onset high myopia (eoHM)/ocular-only Stickler syndrome (ocular-STL) in a large Chinese family. Methods Genomic DNA and clinical data from a four-generation family with eoHM/ocular-STL were collected. Whole-exome sequencing was performed on one affected member in initial screening. Linkage scan based on microsatellite markers was carried out initially from candidate loci associated with autosomal dominant eoHM and Stickler syndrome. Sanger sequencing was used to detect potential variants. The pathogenicity of candidate variants was evaluated using mini genes ex vivo. Results Eight patients and five unaffected members in the family participated in the study, in which the patients had high myopia with other variable ocular phenotypes but without extraocular abnormalities. Whole exome sequencing did not detect any potential pathogenic variant in all genes known to associate with the disease. The eoHM/ocular-STL in the family was mapped to markers around COL2A1 by candidate loci linkage scan, with a maximum lod score of 3.31 for D12S1590 at theta = 0. A novel deep intronic variant, c.86-50C > G in intron 1 of COL2A1, was detected by Sanger sequencing and co-segregated with eoHM/ocular-STL in the family. Ex vivo splicing test using mini genes confirmed that the variant created a new splicing acceptor 49 bp before the canonical splicing site of exon 2, resulted in addition of 49 bp fragment in the transcript (from c.86-49 to c.86-1) and premature termination. Conclusions Linkage study, bioinformatics prediction, and ex vivo transcript analysis suggest a novel deep intronic variant adjacent to 5-prime of exon 2 of COL2A1, affecting exon 2 splicing, as a potential cause of ocular-STL in a large family. To our knowledge, this is the first report of an intronic variant around exon 2 as a cause of ocular-STL while a series of variants in the coding region of exon 2, a dispensable alternative-splicing exon for extraocular tissues, in COL2A1 have been reported to cause Stickler syndrome-related ocular phenotype alone.