Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats

Summary Cells from fetal or neonatal skeleton can synthesize bone-like tissue in vitro. In contrast, formation of bone-like tissue in vitro by cells derived from adult animals has rarely been reported and has not been achieved using cells from bone marrow. We have explored development of bone-like tissue in vitro by bone marrow stromal cells. Marrow stromal cells obtained from 40–43-day-old Wistar rats were grown in primary culture for 7 days and then subcultured for 20–30 days. Cells were cultured in either -minimal essential medium containing 15% fetal bovine serum, antibiotics, and 50 g/ml ascorbic acid, or the above medium supplemented with either 10 mM Na--glycerophosphate, 10^-8 M dexamethasone, or a combination of both. Cultures were examined using phase-contrast microscopy, undemineralized and demineralized tissue histology, histochemistry (for alkaline phosphatase activity), immunohistochemistry (for collagen type, osteonectin, and bone Glaprotein), scanning and transmission electron microscopy, energy dispersive X-ray microanalysis, and X-ray diffraction. Collagenous, mineralized nodules exhibiting morphological and ultrastructural characteristics similar to bone were formed in the cultures, but only in the presence of both -glycerophosphate and dexamethasone. Cells associated with the nodules exhibited alkaline phosphatase activity. The matrix of the nodules was composed predominantly of type-I collagen and both osteonectin and Glaprotein were present. X-ray microanalysis showed the presence of Ca and P, and X-ray diffraction indicated the mineral to be hydroxyapatite. The nodules were also examined for bone morphogenetic protein-like activity. Paired diffusion chambers containing partly demineralized nodules and fetal muscle were implanted intraperitonealy in rats. Induction of cartilage in relation to muscle was observed histologically after 40 days in the chambers. This finding provided further support for the bone-like nature of the nodules. The observations show that bone-like tissue can be synthesized in vitro by cells cultured from young-adult bone marrow, provided that the medium contains both -glycerophosphate and, particularly, dexamethasone.     

Occurrence of osteoblast necroses during ossification of long bone cortices in mouse fetuses

Previous investigations concerned with in vitro osteogenesis and mineralization have revealed some indication of a participation of cell necroses in the course of calcification. These observations were confirmed by in vivo investigations on desmoid ossification in fetal mouse calvariae, where abundant necrotic osteoblasts were found at the mineralization border and in the osteoid. In the present study, ossification of long bone cortices from fetal mice was investigated by use of electron microscopy. Specimens obtained from the collection of the Institute of Anatomy, Free University of Berlin (mouse fetuses, forearm; rat fetuses, forearm) were reinvestigated for control purposes. In all cases, mineralization of osteoid was accompanied by cell necroses. Cell degeneration was characterized by swelling of the endoplasmic reticulum and loss of the plasma membrane resulting in freely distributed vesicular structures. Cell debris was incorporated within the mineral. Initially, cell necroses in the perichondrium occurred in the region surrounding the hypertrophic cartilage and the matrix of which showed spots of endochondral mineralization. Necrotic osteoblasts occurred simultaneously with mineralization of the osteoid. During further ossification of the long bone cortices, the number of necrotic cells increased markedly. In addition to necrotic cells, healthy osteoblasts, osteocytes and perichondral tissue were present, indicating that an artifact can be excluded. The importance of cell necroses in the process of mineralization is as yet unclear. Possibly, the cells act as calcium and/or phosphate stores, which are liberated by cell death to increase the amount of mineral constituents at sites of mineralization.     

遗传性骨病致病基因及相关miRNA的相互作用分析

目的:筛选遗传性骨病相关的致病基因和其相关的mi RNA,并研究两者相互作用关系以及在遗传性骨病中的作用。方法:本研究应用生物信息学的方法,首先应用mirwalk和《国际遗传性骨病分类标准》分析遗传性骨病的致病基因,根据筛选出来的致病基因利用mirwalk的10个预测mirna搜索引擎功能,搜索致病基因的相关mi RNA,并应用excel工作表统计分析mirna的靶向致病基因;再应用Cytoscape软件分析致病基因和相关mirna之间的相互作用关系。结果:本研究中与遗传性骨病密切相关的基因可以分为四类:第一类为成骨不全类基因COL1A1、COL1A2等;第二类为骨密度降低类基因如ACVR1、ALX1等;第三类为骨密度增加类基因如CASR、DMTF1等;第四类为通过作用骨干参与骨密度增加的基因如TGFBR1、MYCN等。其中与成骨不全关系最为密切的mirna是hsa-miR-26b、hsa-miR-19、hsa-miR-200c;与骨密度降低关系最为密切的mirna是hsa-miR-138、hsa-miR-505;与骨密度增加关系最为密切的mirna是hsa-miR-196a、hsa-miR-200b、hsa-miR-19b。结论:mirna可通过调控遗传性骨病致病基因在其病理过程中起到重要作用,提示上述mirna可能是成为遗传性骨病产前筛查和临床药物治疗的新靶点。     

Mepe is expressed during skeletal development and regeneration

Matrix extracellular phosphoglycoprotein (Mepe) is a bone metabolism regulator that is expressed by osteocytes in normal adult bone. Here, we used an immunohistochemical approach to study whether Mepe has a role in murine long bone development and regeneration. Our data showed that Mepe protein was produced by osteoblasts and osteocytes during skeletogenesis, as early as 2 days postnatal. During the healing of non-stabilized tibial fractures, which occurs through endochondral ossification, Mepe expression was first detected in fibroblast-like cells within the callus by 6 days postfracture. By 10 and 14 days postfracture (the hard callus phase of repair), Mepe was expressed within late hypertrophic chondrocytes and osteocytes in the regenerating tissues. Mepe became externalized in osteocyte lacunae during this period. By 28 days postfracture (the remodeling phase of repair), Mepe continued to be robustly expressed in osteocytes of the regenerating bone. We compared the Mepe expression profile with that of alkaline phosphatase, a marker of bone mineralization. We found that both Mepe and alkaline phosphatase increased during the hard callus phase of repair. In the remodeling phase of repair, Mepe expression levels remained high while alkaline phosphatase activity decreased. We also examined Mepe expression during cortical bone defect healing, which occurs through intramembranous ossification. Mepe immunostaining was found within fibroblast-like cells, osteoblasts, and osteocytes in the regenerating bone, through 5 to 21 days postsurgery. Thus, Mepe appears to play a role in both long bone regeneration and the latter stages of skeletogenesis.     

Osteogenic differentiation of the mesenchymal progenitor cells, Kusa is suppressed by Notch signaling

Notch receptor plays a crucial role in proliferation and differentiation of many cell types. To elucidate the function of Notch signaling in osteogenesis, we transfected the constitutively active Notch1 (Notch intracellular domain, NICD) into two different osteoblastic mesenchymal cell lines, KusaA and KusaO, and examined the changes of their osteogenic potentials. In NICD stable transformants (KusaA(NICD) and KusaO(NICD)), osteogenic properties including alkaline phosphatase activity, expression of osteocalcin and type I collagen, and in vitro calcification were suppressed. Transient transfection of NICD attenuated the promoter activities of Cbfa1 and Ose2 element. KusaA was capable of forming trabecular bone-like tissues when injected into mouse abdomen, but this in vivo bone forming activity was significantly suppressed in KusaA(NICD). Osteoclasts were induced in the KusaA-derived bone-like tissues, but lacked in the KusaA(NICD)-derived tissues. These results suggest that Notch signaling suppresses the osteoblastic differentiation of mesenchymal progenitor cells.     

Enhancement of bone repair with a helper-dependent adenoviral transfer of bone morphogenetic protein-2

Regional gene therapy, which involves the delivery of growth factors to a specific anatomic site, has the potential to enhance bone formation in clinical application. Helper-dependent adenoviral vectors, which have deleted all of the viral coding regions, have been shown to be safe and highly efficient with long-lasting transgene expression. In this study, we constructed a helper-dependent adenoviral vector producing bone morphogenetic protein-2 (AdHDBMP-2). The AdHDBMP-2 increased the alkaline phosphatase activity of W-20-17 cells in vitro. In addition, when AdHDBMP-2 infected rat bone marrow cells were implanted into the hindlimbs of SCID mice, orthotopic bone formation was shown at 2 weeks. To our knowledge, this is the first study to demonstrate bone formation with the helper-dependent adenoviral vector with the BMP-2 expression cassette. This type of gene therapy vector could prove to be highly useful for bone augmentation in patients with bone loss associated with trauma, revision total joint arthroplasty, or cancer.