Combined effects of surface morphology and mechanical straining magnitudes on the differentiation of mesenchymal stem cells without using biochemical reagents.

Existing studies examining the control of mesenchymal stem cell (MSC) differentiation into desired cell types have used a variety of biochemical reagents such as growth factors despite possible side effects. Recently, the roles of biomimetic microphysical environments have drawn much attention in this field. We studied MSC differentiation and changes in gene expression in relation to osteoblast-like cell and smooth muscle-like cell type resulting from various microphysical environments, including differing magnitudes of tensile strain and substrate geometries for 8 days. In addition, we also investigated the residual effects of those selected microphysical environment factors on the differentiation by ceasing those factors for 3 days. The results of this study showed the effects of the strain magnitudes and surface geometries. However, the genes which are related to the same cell type showed different responses depending on the changes in strain magnitude and surface geometry. Also, different responses were observed three days after the straining was stopped. These data confirm that controlling microenvironments so that they mimic those in vivo contributes to the differentiation of MSCs into specific cell types. And duration of straining engagement was also found to play important roles along with surface geometry.

A hyaluronate-atelocollagen/beta-tricalcium phosphate-hydroxyapatite biphasic scaffold for the repair of osteochondral defects: a porcine study.

The authors had devised a novel biphasic scaffold combining hyaluronic acid and atelocallagen for the chondral phase and combining hydroxyapatite and beta-tricalcium phosphate for the osseous phase. Sixty-four osteochondral defects were created in the knee joints of 16 minipigs to evaluate the effectiveness of this scaffold for repairing cartilage in a large animal model. The defects were divided into five groups according to their treatment: filling with a cell/biphasic scaffold composite (Group I, 16 defects); implanting only the biphasic scaffold (Group II, 16 defects); placing the removed osteochondral fragments back into the defect (Group IIIa, 8 defects); autologous chondrocyte implantation (Group IIIb, 8 defects); leaving the defects empty (Group IV, the negative control). After 5 months, the International Cartilage Repair Society Macroscopic Score was similar in Group I (9.0), Group II (9.1), and Group IIIa (9.1), followed by Group IIIb (7.4) and Group IV (6.2). Except for three defects noted in Group IV, all the defects were filled with cartilaginous or fibrous tissue depending on the groups. The junction to the adjacent native cartilage was detectable in all the groups of minipigs. Microscopically, Group II had the highest score from the International Cartilage Repair Society Visual Histological Assessment Scale. The indentation study showed that the maximum loads and time constant of Group I, II, and IIIa defects were comparable to that of native cartilage, whereas the equilibrium loads of these groups were slightly greater than that of native cartilage. In conclusion, our results suggest that a biphasic osteochondral scaffold with a chondral phase, consisting of hyaluronate and atelocollagen, and an osseous phase, consisting of hydroxyapatite and beta-tricalcium phosphate, is effective for repairing osteochondral defects in a large animal model.

Enhanced differentiation of mesenchymal stem cells into NP-like cells via 3D co-culturing with mechanical stimulation.

This study proposes a three-dimensional co-culturing system of mesenchymal stem cells (MSCs) and nucleus pulposus (NP) cells from New Zealand white male rabbits to differentiate MSCs into NP-like cells. The preferable ratio of MSCs to NP cells and the effects of mechanical stimulation were investigated without biochemical reagents. The preferable ratio was investigated without mechanical stimulation using five groups: Group I (MSC control); Group II (NP cell control); and Groups III, IV, and V, for which the ratios of NP cells to MSCs were 1:1, 1:2, and 2:1, respectively. During culture for 10 days without stimulation, the proliferation of MSCs did not increase after day 4. NP cells proliferated more when co-cultured as in Group V. However, the degree of differentiation of MSCs increased significantly in Group V. The differentiation of NP cells decreased gradually over time. When mechanical stimulation was applied to Groups I, II, and V, it contributed to the differentiation of MSCs into NP-like cells, as well as to that of NP cells, but did not contribute to the proliferation of either cell type. The contribution of mechanical stimulation to differentiation was also confirmed by RT-PCR.

Development of a molecular marker specific to a novel CMS line in radish (Raphanus sativus L.).

In this study, we have investigated the cytoplasmic male sterility (CMS) of a novel male sterile radish line, designated NWB CMS. The NWB CMS was crossed with 16 fertile breeding lines, and all the progenies were completely male sterile. The degree of male sterility exhibited by NWB CMS is more than Ogura CMS from the Cruciferae family. The NWB CMS was found to induce 100% male sterility when crossed with all the tested breeding lines, whereas the Ogura CMS did not induce male sterility with any of the breeding lines. PCR analysis revealed that the molecular factor that influenced Ogura CMS, the orf138 gene, was absent in the NWB CMS line, and that the orf138 gene was not also expressed in this CMS line. In order to identify the cytoplasmic factors that confer male sterility in the NWB CMS line, we carried out RFLP analyses with 32 mitochondrial genes, all of which were used as probes. Fourteen genes exhibited polymorphisms between the NWB CMS line and other radish cultivars. Based on these RFLP data, intergenic primers were developed in order to amplify the intergenic regions between the polymorphic genes. Among these, a primer pair at the 3' region of the atp6 gene (5'-cgcttggactatgctatgtatga-3') and the 5' region of the nad3 gene (5'-tcatagagaaatccaatcgtcaa-3') produced a 2 kbp DNA fragment as a result of PCR. This DNA fragment was found to be specific to NWB CMS and was not present in other CMS types. It appears that this fragment could be used as a DNA marker to select NWB CMS line in a radish-breeding program.