Active creatine kinase is present in matrix vesicles isolated from femurs of chicken embryo: Implications for bone mineralization

Proteomic analysis of matrix vesicles (MVs) isolated from 17-day-old chicken embryo femurs revealed the presence of creatine kinase. In this report we identified the enzyme functionally and suggest that the enzyme may participate in the synthesis of ATP from ADP and phosphocreatine within the lumen of these organelles. Then, ATP is converted by nucleotide hydrolyzing enzymes such as Na⁺, K⁺-ATPase, protein kinase C, or alkaline phosphatase to yield inorganic phosphate (P_i), a substrate for mineralization. Alternatively, ATP can be hydrolyzed by a nucleoside triphosphate pyrophosphatase phosphodiesterase 1 producing inorganic pyrophosphate (PP_i), a mineralization inhibitor. In addition, immunochemical evidence indicated that VDAC 2 is present in MVs that may serve as a transporter of nucleotides from the extracellular matrix. We discussed the implications of ATP production and hydrolysis by MVs as regulatory mechanisms for mineralization.     

Proteome analysis of matrix vesicles isolated from femurs of chicken embryo

Matrix vesicles (MVs) are extracellular organelles that initiate mineral formation, accumulating inorganic phosphate (P(i)) and calcium leading to the formation of hydroxyapatite (HA) crystals, the main mineral component of bones. MVs are produced during bone formation, as well as during the endochondral calcification of cartilage. MVs are released into the extracellular matrix from osseous cells such as osteoblasts and hypertrophic chondrocytes. In this report, using 1-D SDS-PAGE, in-gel tryptic digestion and an LC-MS-MS/MS protein identification protocol, we characterized the proteome of MVs isolated from chicken embryo (Gallus gallus) bones and cartilage. We identified 126 gene products, including proteins related to the extracellular matrix and ion transport, as well as enzymes, cytoskeletal, and regulatory proteins. Among the proteins recognized for the first time in MVs were aquaporin 1, annexin A1 (AnxA1), AnxA11, glycoprotein HT7, G(i) protein alpha2, and scavenger receptor type B. The pathways for targeting the identified proteins into MVs and their particular functions in the biomineralization process are discussed. Obtaining a knowledge of the functions and roles of these proteins during embryonic mineralization is a prerequisite for the overall understanding of the initial mineral formation mechanisms.     

Implantation in vitro: co-culture of rat blastocyst and epithelial cell vesicles

Implantation of blastocysts involves conversion of maternal and embryonic cell surfaces from a nonadhesive to an adhesive state in response to the internally driven developmental program or to externally generated factors. However, the intricacies of the cellular and subcellular changes that promote the attachment are not known, because these changes are difficult to determine in situ because of the nonaccessibility of the site. To overcome this, an in vitro model of implantation was developed by co-culturing rat blastocysts and uterine epithelial cells of the same gestational age (day 5 postcoitum; plug day as day 1) in drops hanging from the lid of a Petri dish. The system was used to study the changes on the surface membranes of the cells of the trophectoderm and uterine epithelium and to evaluate the antiadhesive activity of the newly designed test substances. The isolated epithelial cell vesicles were co-cultured with zona-free blastocysts in the microdrops (40–50 µl) hanging from the lid of a 60-mm Petri dish. The lid was placed over the lower dish, which was presaturated with the medium. The culture was examined 48 h later to determine the site of adhesion of epithelial cell vesicles with the trophoblasts lining the blastocyst. The cell-cell adhesion was monitored on a computerized image analyzer. To validate the adhesion of blastocysts and epithelial cell vesicles in co-culture, the expression of a cell adhesion molecule, uvomorulin, was studied using immunocytochemical technique after incubating with antiuvomorulin antibody. Intense staining was noted on the membrane surfaces at the site of attachment of the blastocyst and cell vesicles.The authors express their sincere thanks to the Ministry of Health and Family Welfare, Government of India, for their financial support     

Survival and proliferation of rat gonocytes in vitro

Quiescent gonocytes were isolated from fetal testes of rat 18-day post coitum and cultured alone or on monolayers of somatic cells from different origins. The gonocytes specifically adhered to Sertoli cells, isolated from 21 to 23-day-old rat testes; this adherence was necessary for their survival in vitro. Addition of follicle-stimulating hormone and testosterone to these cultures did not increase the viability of the gonocytes. Serum was found to be deleterious to the germ cells. Electron-microscopic examination of Sertoli-cell-gonocyte co-cultures revealed the presence of numerous adhesion plaques between these cells, indicating that Sertoli cells and gonocytes are able to communicate in vitro. Gonocytes, in co-culture with Sertoli cells, were viable for at least 9 days. The gonocytes did not spontaneously resume proliferation. The simple culture system described in the present paper should be useful in studying the nature of the factors that are responsible for sending the quiescent gonocytes into the cell cylce and for stimulating the formation of A spermatogonia, a process characterizing the start of spermatogenesis.     

小鼠骨髓源树突状细胞体外诱导培养及初步鉴定

用重组小鼠粒细胞-巨噬细胞集落刺激因子（rmGM-CSF）和重组小鼠白细胞介素4（rmIL-4）体外诱导小鼠骨髓细胞分化为树突状细胞,进行形态学变化观察,分析细胞表面分子,刺激T细胞增殖,探讨小鼠骨髓源树突状细胞（BMDC）体外诱导培养并进行初步鉴定。体外培养9d后BMDC可达80%以上,光镜下可见典型的树突状细胞形态。清楚表达成熟期主要表面标志物,可显著刺激同种异体混合淋巴细胞增殖。获得了较高纯度的BMDC,避免了使用传统磁珠分离方法所带来的成本高,操作复杂,产出率低的弊端,为研究BMDC功能以及运用开展下游实验提供材料。     

Culture and neural differentiation of rat bone marrow mesenchymal stem cells in vitro

Adult bone marrow mesenchymal stem cells (MSCs) can differentiate into several types of mesenchymal cells, including osteocytes, chondrocytes, and adipocytes, but can also differentiate into non-mesenchymal cells, such as neural cells, under appropriate experimental conditions. Until now, many protocols for inducing neuro-differentiation in MSCs in vitro have been reported. But due to the differences in MSCs' isolation and culture conditions, the results of previous studies lacked consistency and comparability. In this study, we induced differentiation into neural phenotype in the same MSCs population by three different treatments: beta-mercaptoethanol, serum-free medium and co-cultivation with fetal mouse brain astrocytes. In all of the three treatments, MSCs could express neural markers such as NeuN or GFAP, associating with remarkable morphological modifications. But these treatments led to neural phenotype in a non-identical manner. In serum-free medium, MSCs mainly differentiated into neuron-like cells, expressing neuronal marker NeuN, and BME can promote this process. Differently, after co-culturing with astrocytes, MSCs leaned to differentiate into GFAP(+) cells. These data confirmed that MSCs can exhibit plastic neuro-differentiational potential in vitro, depending on the protocols of inducement.     

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.     

In vitro decidualization of rat endometrial stromal cells

The induction of the decidualization of endometrial stromal cells is possible in an in vitro cell culture system. However, thus far, methods differ according to species or cell type, and a more stable or universal system has not yet been developed. The purpose of the present study has been to establish an in vitro decidualization system in primary cultured rat endometrial stromal cells (RES). The RES were treated with medroxyprogesterone acetate and dibutyryl-cyclic adenosine monophosphate (MPA treatment), estradiol and progesterone, or arachidonic acid. After 24 h of treatment, cells responded to all of the stimulations by expressing desmin mRNA. However, decidual/trophoblast prolactin-related protein (dPRP) mRNA was only expressed in the MPA-treated cells. Desmin and dPRP mRNA were not expressed after MPA treatment of the RES derived from immature rat uteri. However, mRNA from both desmin and dPRP were expressed in RES derived from gonadotrophin-injected immature rats. The expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 mRNA did not change after the decidual treatment of RES examined by real-time polymerase chain reaction. However, the results of gelatin zymography showed that the active forms of MMP-2 and MMP-9 significantly increased after in vitro decidualization (P < 0.05). We conclude that MPA treatment is the most effective method for stimulating decidualization in RES. Use of this system has revealed that sexual maturation and gonadotrophins are important for RES with regard to decidualization. Furthermore, the activity of MMP-2 and MMP-9 might increase during decidualization without a corresponding increase of the expression of these genes. This research was supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (JSPS; no. 18580282, to N. Yamauchi).     

Extracellular matrix vesicles in rat bone after parathyroidectomy

Summary The enzymatic activity of bone matrix vesicles from parathyroidectomized rats was determined and compared to the activity of vesicles from sham operated and normal animals. The vesicles were isolated from the alveolar bone by collagenase digestion and differential centrifugation and further purified on a discontinuous sucrose density gradient. The amount of extractable protein and the activity of alkaline phosphatase, acid phosphatase, and ATPase in the vesicle fractions thus obtained did not differ significantly from the values characteristic of preparations from control rats. It may therefore be suggested that parathyroid hormone depletion and the associated hypocalcemia have no significant effect on the occurrence and phosphatase activity of bone matrix vesicles.     

体外培养成骨细胞的影响因素

成骨细胞是骨形成和骨代谢的核心部分,成骨细胞体外培养是研究骨代谢和成骨机制的重要手段。本从物理因素,微量元素,生长因子和激素等四个方面综述了体外培养成骨细胞的影响因素,以期有助于研究有效的体外培养成骨细胞的方法应用于组织工程学的研究。     

大鼠表皮细胞的体外培养及纯化方法

根据表皮细胞和成纤维细胞对胰蛋白酶敏感性、贴壁时间及要求不同的,采用胰蛋白酶消化法和反复贴壁法相结合,能有效清除成纤维细胞的混合生长,获得纯化的表皮细胞。本方法经济、简便、实用。     

Long-chain polyphosphate in osteoblast matrix vesicles: Enrichment and inhibition of mineralization

Background

Inorganic polyphosphate (polyP) is a fundamental and ubiquitous molecule in prokaryotes and eukaryotes. PolyP has been found in mammalian tissues with particularly high levels of long-chain polyP in bone and cartilage where critical questions remain as to its localization and function. Here, we investigated polyP presence and function in osteoblast-like SaOS-2 cells and cell-derived matrix vesicles (MVs), the initial sites of bone mineral formation.

The relative magnitudes of endothelial force generation and matrix stiffness modulate capillary morphogenesis in vitro

When suspended in collagen gels, endothelial cells elongate and form capillary-like networks containing lumens. Human blood outgrowth endothelial cells (HBOEC) suspended in relatively rigid 3 mg/ml floating collagen gels, formed in vivo-like, thin, branched multi-cellular structures with small, thick-walled lumens, while human umbilical vein endothelial cells (HUVEC) formed fewer multi-cellular structures, had a spread appearance, and had larger lumens. HBOEC exert more traction on collagen gels than HUVEC as evidenced by greater contraction of floating gels. When the stiffness of floating gels was decreased by decreasing the collagen concentration from 3 to 1.5 mg/ml, HUVEC contracted gels more and formed thin, multi-cellular structures with small lumens, similar in appearance to HBOEC in floating 3 mg/ml gels. In contrast to floating gels, traction forces exerted by cells in mechanically constrained gels encounter considerable resistance. In constrained collagen gels (3 mg/ml), both cell types appeared spread, formed structures with fewer cells, had larger, thinner-walled lumens than in floating gels, and showed prominent actin stress fibers, not seen in floating gels. These results suggest that the relative magnitudes of cellular force generation and apparent matrix stiffness modulate capillary morphogenesis in vitro and that this balance may play a role in regulating angiogenesis in vivo.     

No evidence of mineralization abnormalities in iliac bone of premenopausal women with type 2 diabetes mellitus

Objectives:Patients with type-2 diabetes mellitus (T2DM) have increased risk for bone fractures which points towards impaired bone quality.Methods:We measured bone mineralization density distribution (BMDD) and osteocyte lacunae section (OLS) characteristics based on quantitative backscattered electron images of transiliac biopsy samples from n=26 premenopausal women with T2DM. Outcomes were compared to those from reference cohorts as well as between T2DM subgroups defined by clinical characteristics.Results:Comparison to references did not reveal any differences in BMDD (all p>0.05) but a lowered OLS-density in cancellous bone in T2DM (-14.9%, p<0.001). Neither BMDD nor OLS-characteristics differed in T2DM subgroups defined by HbA1c (<7% versus >7%). The average degree of bone mineralization (CaMean) was higher (0.44 wt%Ca in T2DM, 0.30 wt%Ca in reference) and consistently the calcium concentration between the tetracycline double labels (CaYoung) was higher (0.76 wt%Ca, all p<0.001) in cancellous versus cortical bone.Conclusions:Our findings suggest that bone matrix mineralization was neither affected by the presence nor by the glycemic control of T2DM in our study cohort. The intra-individual differences between cancellous and cortical bone mineralization gave evidence for differences in the time course of the early mineralization process in these compartments in general.     

Heritable variability induced by the in vitro culture and genetic improvement of cultivated plants

Abstract

Genetic variability is found among plants derived from in vitro cultures of somatic cells. A number of different factors, such as the pre-existing genetic variation developed in vivo during tissue differentiation, the variation induced during the in vitro culture and also the selection for specific genotypes during plant regeneration, are considered as possible causes of the phenomenon.

The nature of the genetic changes induced in somaclones (variation in chromosome number, gross and cryptic chromosomal rearrangements, transposition of genetic elements, gene amplification and somatic gene rearrangements) is also discussed.     

束状刺盘孢的体外培养条件研究

目的对束状刺盘孢体外培养,观察形态特征。方法将束状刺盘孢接种在马铃薯葡萄糖琼脂培养基(PDA)中分别置于4℃、28℃、35℃和37℃下培养2周,观察生长情况;选取生长最好的菌株分别接种在沙堡弱培养基(SDA)、PDA、察氏培养基(CPA)、胡萝卜琼脂培养基(CDA)和玉米粉培养基(CMA)中,同一温度下培养2周,观察菌落形态及镜下形态。结果菌株在28℃条件下生长最快、菌落发育饱满、产生灰色色素;菌株在五种培养基中生长快慢依次是PDA>CDA>CMA>SDA>CPA,菌落在PDA、CDA和CMA中呈鼠灰色,SDA中呈白色和棕色,CPA中呈白色和鼠灰色,SDA和CPA中未见分生孢子和刚毛产生。结论在PDA、CDA和CMA中28℃条件下,较适合束状刺盘孢生长。     

Reduced mitotic activity at the periphery of human embryonic stem cell colonies cultured in vitro with mitotically-inactivated murine embryonic fibroblast feeder cells

This study attempted to investigate whether different levels of mitotic activity exist within different physical regions of a human embryonic stem (hES) cell colony. Incorporation of 5-bromo-2-deoxyuridine (BrdU) within newly-synthesized DNA, followed by immunocytochemical staining was used as a means of detecting mitotically-active cells within hES colonies. The results showed rather surprisingly that the highest levels of mitotic activity are primarily concentrated within the central regions of hES colonies, whereas the peripheral regions exhibited reduced levels of cellular proliferation. Two hypothetical mechanisms are therefore proposed for hES colony growth and expansion. Firstly, it is envisaged that the less mitotically-active hES cells at the periphery of the colony are continually migrating outwards, thereby providing space for newly-divided daughter cells within the more mitotically-active central region of the hES colony. Secondly, it is proposed that the newly-divided hES cells within the central region of the colony somehow migrate to the outer periphery. This could possibly explain why the periphery of hES colonies are less mitotically-active, since there would obviously be an extended time-lag before newly-divided daughter cells are ready again for the next cell division. Further investigations need to be carried out to characterize the atypical mechanisms by which hES colonies grow and expand in size.     

In vitro differentiation and mineralization of cartilaginous nodules from enzymatically released rat nasal cartilage cells

Nasal cartilage cells from 21-day-old rat fetuses were cultured at high density in the presence of ascorbic acid and β-glycerophosphate over a 12-day period. Immediately after plating, the cells exhibited a fibroblastic morphology, lost their chondrocyte phenotype and expressed type I collagen. On day 3, clusters of enlarged polygonal cells were found. These cell clusters synthetised type II collagen and formed an alcian-blue-positive matrix. The following days, a progressive increase in the number of cells positive for type 11 collagen was noted and, on day 8, typical cartilaginous nodules were formed. These nodules increased in size and number, spreading outward, laying down a dense matrix which mineralized. Light and electron microscopy observations of cross-sections of nodules confirmed the cartilaginous nature of this tissue formed in vitro with typical chondrocytes embedded in a hyaline matrix. Furthermore, at the electron microscopic level, matrix vesicles were seen in extracellular matrix associated with the initiation of mineralization. Typical rod-like crystals were present in the intercellular spaces along the collagen fibers. These results indicated that in a specific environment, dedifferentiated chondrocytes were able to redifferentiate and to form nodular structures with morphological ultrastructure of calcified cartilage observed in vivo.