Enhanced spreading, migration and osteodifferentiation of HBMSCs on macroporous CS-Ta - A biocompatible macroporous coating for hard tissue repair.

Macroporous tantalum (Ta) coating was produced on titanium alloy implant for bone repair by cold spray (CS) technology, which is a promising technology for oxygen sensitive materials. The surface characteristics as well as in vitro cytocompatibility were systematically evaluated. The results showed that a rough and macroporous CS-Ta coating was formed on the Ti6Al4V (TC4) alloy surfaces. The surface roughness showed a significant enhancement from 17.06 µm (CS-Ta-S), 27.48 µm (CS-Ta-M) to 39.21 µm (CS-Ta-L) with the increase of the average pore diameter of CS-Ta coatings from 138.25 µm, 198.25 µm to 355.56 µm. In vitro results showed that macroporous CS-Ta structure with tantalum pentoxide (Ta2O5) was more favorable to induce human bone marrow derived mesenchymal stem cells (HBMSCs) spreading, migration and osteodifferentiation than TC4. Compared with the micro-scaled structure outside the macropores, the surface micro-nano structure inside the macropores was more favorable to promote osteodifferentiation with enhanced alkaline phosphatase (ALP) activity and extracellular matrix (ECM) mineralization. In particular, CS-Ta-L with the largest pore size showed significantly enhanced integrin-α5 expression, cell migration, ALP activity, ECM mineralization as well as osteogenic-related genes including ALP, osteopontin (OPN) and osteocalcin (OCN) expression. Our results indicated that macroporous Ta coatings by CS, especially CS-Ta-L, may be promising for hard tissue repairs.

Bid and Bax are involved in granulosa cell apoptosis during follicular atresia in porcine ovaries.

More than 99% of follicles undergo "atresia" during follicular development and growth. Follicular atresia is predominantly regulated by granulosa cell apoptosis. However, the intracellular signaling pathway of apoptosis in granulosa cells has not been revealed. In the present study, we examined changes in the expression of BH3-interacting domain death agonist (Bid) and Bcl-2-associated X protein (Bax), which are considered to promote the cell death ligand/receptor-mediated process in mitochondrion-dependent type II apoptosis, in porcine granulosa cells during atresia. Levels of mRNA and protein of Bid and Bax were determined by the reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting techniques, respectively. Levels of Bid and Bax mRNA and protein were markedly increased in granulosa cells of early atretic follicles compared with those of healthy follicles. In situ hybridization and immunohistochemical staining revealed that mRNA and protein of Bid and Bax were present in the granulosa cells, though only traces were found in healthy follicles; however, strong staining was noted in atretic follicles. These results indicate that Bid and Bax appear to be signal transduction factors in granulosa cells during follicular atresia and appear to play proapoptotic roles and confirm that the porcine granulosa cell is a mitochondrion-dependent type II apoptotic cell.

Changes in the expression of decoy receptor 3 in granulosa cells during follicular atresia in porcine ovaries.

During follicular development in mammalian ovaries, the majority of follicles undergo atresia. One of the characteristics of this process is apoptotic cell death in granulosa cells. Several death ligands and receptors, including Fas ligand (FasL) and Fas, have been detected in ovarian follicles and also demonstrated to be capable of inducing apoptosis in follicular cells. Decoy receptor 3 (DcR3) competes with Fas to bind FasL but lacks intracellular death domains, thus inhibiting the induction of apoptosis by the FasL/Fas system. In the present study, we examined the expression of putative porcine DcR3 (pDcR3) mRNA in porcine ovarian follicles. Total RNA was extracted from granulosa cells and thecal layer cells of tertiary follicles at healthy, early atretic and progressed atretic stages, and the expression of pDcR3 mRNA was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR). The nucleic acid sequence in the coding region had 80% homology to that of human DcR3, and the deduced amino acid sequence was 73% identical to that of human DcR3. In an in situ hybridization experiment, pDcR3 mRNA expression was confirmed in granulosa and thecal layers, in both healthy and atretic follicles. Quantitative real time RT-PCR analysis showed that the expression of pDcR3 mRNA was weaker in granulosa cells of atretic follicles than those of healthy follicles. No notable changes were seen in the thecal layer cells. These results suggest that DcR3 plays a significant role in the regulation of apoptosis in granulosa cells, but not in thecal layer cells, during atresia.