In the present study the in vivo mechanism of calcium-phosphate (CaP) ceramic degradation has been investigated by means of transmission electron microscopy. The results revealed osteoclast-mediated degradation of hydroxyapatite ceramic implanted into sheep bone by simultaneous resorption and phagocytosis. After 6 weeks of implantation, osteoclasts were localized immediately beneath the ceramic surface. They had formed resorption lacunae and exhibited typical ultrastructural features, such as the ruffled border, the clear zone, and the dorsal microvilli. Their resorption capacity also had become evident by alterations of the electron density and the shape of the CaP crystals localized within the acidic microenvironment of the ruffled border. Moreover, the osteoclasts simultaneously were capable of phagocytosing the resorbed CaP crystals. The formation of endophagosomes was performed (1) by the uptake of particles into large intracellular vacuoles, which were generated by deep invagination of the membranes of the osteoclastic ruffled border, and (2) by the encircling of particles due to the development of pseudopodia-like plasmaprotrusions of the ruffled border. The formation of endophagosomes was followed by the in situ fragmentation of the inclusion material, which subsequently was released into the extracellular space and phagocytosed by macrophages.
Adipose-derived stromal cells (ADSCs) are multipotent cells which, in the presence of appropriate stimuli, can differentiate into various lineages such as the osteogenic, adipogenic and chondrogenic. In this study, we investigated the effect of transforming growth factor beta 1 (TGF-β1) in comparison to hydrolyzed fish collagen in terms of the chondrogenic differentiation potential of ADSCs. ADSCs were isolated from subcutaneous fat of horses by liposuction. Chondrogenesis was investigated using a pellet culture system. The differentiation medium was either supplemented with TGF-β1 (5 ng/ml) or fish collagen (0.5 mg/ml) for a 3 week period. After the 3 weeks in vitro differentiation, RT-PCR and histological staining for proteoglycan synthesis and type II collagen were performed to evaluate the degree of chondrogenic differentiation and the formation of cartilaginous extracellular matrix (ECM). The differentiation of ADSCs induced by TGF-β1 showed a high expression of glycosaminoglycan (GAG). Histological analysis of cultures stimulated by hydrolyzed fish collagen demonstrated an even higher GAG expression than cultures stimulated under standard conditions by TGF-β1. The expression of cartilage-specific type II collagen and Sox9 was about the same in both stimulated cultures. In this study, chondrogenesis was as effectively induced by hydrolyzed fish collagen as it was successfully induced by TGF-β1. These findings demonstrated that hydrolyzed fish collagen alone has the potential to induce and maintain ADSCs-derived chondrogenesis. These results support the application of ADSCs in equine veterinary tissue engineering, especially for cartilage repair.
DNA methylation catalyzed by DNA methyltransferases (DNMTs) and histone deacetylation catalyzed by histone deacetylases (HDACs) play an important role for the regulation of gene expression during carcinogenesis and spermatogenesis. We therefore studied the cell-specific expression of DNMT1 and HDAC1 for the first time in human testicular cancer and impaired human spermatogenesis. During normal spermatogenesis, DNMT1 and HDAC1 were colocalized in nuclei of spermatogonia. While HDAC1 was additionally present in nuclei of Sertoli cells, DNMT1 was restricted to germ cells exhibiting a different expression pattern of mRNA (in pachytene spermatocytes and round spermatids) and protein (in round spermatids). Interestingly, in infertile patients revealing round spermatid maturation arrest, round spermatids lack DNMT1 protein, while pachytene spermatocytes became immunopositive for DNMT1. In contrast, no changes in the expression pattern could be observed for HDAC1. This holds true also in testicular tumors, where HDAC1 has been demonstrated in embryonal carcinoma, seminoma and teratoma. Interestingly, DNMT1 was not expressed in seminoma, but upregulated in embryonal carcinoma.
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