Human bone marrow stromal cells express an osteoblastic phenotype in culture

Summary This study reports the selection and characterization of osteogenic precursors from human bone marrow which were isolated by two “clonings” and successive subculturing. These cell lines express alkaline phosphatase activity. Gel electrophoresis of [³H]-proline labeled cultures showed that the cloned cells produce only type I collagen. They synthetize osteocalcin and osteonectin. They respond to 1,25 dihydroxy vitamin D₃ by increasing osteocalcin synthesis and secretion, and to parathyroid hormone by increasing cyclic AMP synthesis. After the third subculture in the absence of β-glycerophosphate, these cell lines formed lots of clusters which exhibit high alkaline phosphatase activity and positive von Kossa staining. X-ray energy spectrum shows that these cells are surrounded by “budding” structures containing calcium and phosphorus with a ratio Ca:P identical to those of pure hydroxyapatite. This process was associated with⁴⁵Ca uptake into the cells. All these data support the selection of osteogenic cells which may be of considerable clinical importance.     

Effect of 1,25(OH)2D3 on bone morphogenetic protein-3 mRNA expression

Bone morphogenetic proteins (BMPs) are members to the transforming growth factor-beta superfamily. They induce ectopic bone formation in rat and are pleiotropic initiators of inducible osteogenic precursor cells. A lot of reports have studied the presence of BMPs and their effects on bone marker expression in many different cell lines, however none describe the regulation of BMP3 by different factors and expression conditions. When a human bone marrow stromal cell (HBMSC) culture was treated simultaneously with 1,25(OH)2D3 (10(-8) M) and BMP3 (2.5 ng/ml), the total osteocalcin content in the cell layer and in the culture medium was higher than when the culture was treated with either factor alone (162%). To elucidate this synergistic activity, Northern blot analysis was done to study the effect of 1,25(OH)2D3 on BMP3 mRNA expression. Several human cell lines (MNNG, U-2OS, MG-63, KHOS, TE85, HOS) and HBMSC were treated by 1,25(OH)2D3 (10(-8) M for 24 h). Purified mRNA from treated and untreated cells were denatured using glyoxal and dimethylsulfoxide, and were fractionated on a 1% agarose gel. After electrophoresis, RNA were blotted onto a nylon membrane and incubated with 32P-labeled BMP3 and GAPDH riboprobes. Northern blot analysis revealed that, the BMP3 mRNA level was increased in a few cell lines (MG-63, HBMSC, HOS) after the addition of 1,25(OH)2D3 when compared to the untreated cells (127%+/-1; 130.5%+/-19.5; 207%+/-14). An higher stimulation was observed in HBMSC primary culture when compared to differentiated HBMSC. In view of these results, we now investigate the following hypothesis: does the BMP3 promoter exhibit the vitamin D receptor response like the osteocalcin gene?     

Caution regarding the combined effects of extracellular matrices and nutrient media on cultured endothelial cells

To study the influence of smooth muscle cells (SMC) on endothelial cells (EC), different co-culture designs are available, including EC seeding on SMC extracellular matrix (ECM). We explored human umbilical vein endothelial cell (HUVEC) adhesion and proliferation on either in situ or coated ECM, elaborated by HUVECs or human arterial smooth muscle cells (HUASMCs), in the presence of different nutrient media containing varying amounts of fetal calf serum. Coating wells with HUVEC or HUASMC ECMs did not improve HUVEC adhesion 1 h after cell seeding, compared with uncoated wells. HUVEC adhesion on in situ HUVEC-ECM and HUASMC-ECM was significantly increased compared with uncoated wells. The substratum upon which cells are maintained was found to play a crucial role, in conjunction with the medium to which HUVECs are exposed for their proliferative response. These results stress the importance of selecting media in relation to the particular substratum, in order to avoid misinterpretation of data.     

Effect of human endothelial cells on human bone marrow stromal cell phenotype: role of VEGF?

Angiogenesis is a tightly regulated process involved in growth, repair, and bone remodeling. Several studies have shown that there is a reciprocal regulation and functional relationship between endothelial cells and osteoblast-like cells during osteogenesis, where systemic hormones and paracrine growth factors play an active role. Angiogenesis is induced by a variety of growth factors; among them vascular endothelial growth factor (VEGF) may be an important mediator for the angiogenic process involved in bone physiology. We studied the VEGF effect on osteoblast progenitor cells (Human Bone Marrow Stromal Cells: HBMSE) cultured alone or associated with endothelial cells (Human Umbilical Vein Endothelial Cells: HUVEC) in different co-culture models (co-culture with or without direct contact, conditioned medium), to determine the influence of VEGF on these cells and on their relationship. In agreement with other studies, we show that HBMSC express and synthesize VEGF, HUVEC conditioned medium has a proliferative effect on them, and early osteoblastic marker (Alkaline phosphatase activity) levels increase when these cells are co-cultured with HUVEC only in direct contact. However, unlike previous studies, we did not find that VEGF increased these processes. These results suggest that the intercommunication between endothelial cells and osteoblastic-like cells requires not only diffusible factors, but also involving cell membrane proteins.     

The role of vascular actors in two dimensional dialogue of human bone marrow stromal cell and endothelial cell for inducing self-assembled network

Angiogenesis is very important for vascularized tissue engineering. In this study, we found that a two-dimensional co-culture of human bone marrow stromal cell (HBMSC) and human umbical vein endothelial cell (HUVEC) is able to stimulate the migration of co-cultured HUVEC and induce self-assembled network formation. During this process, expression of vascular endothelial growth factor (VEGF₁₆₅) was upregulated in co-cultured HBMSC. Meanwhile, VEGF₁₆₅-receptor2 (KDR) and urokinase-type plasminogen activator (uPA) were upregulated in co-cultured HUVEC. Functional studies show that neutralization of VEGF₁₆₅ blocked the migration and the rearrangement of the cells and downregulated the expression of uPA and its receptor. Blocking of vascular endothelial-cadherin (VE-cad) did not affect the migration of co-cultured HUVEC but suppressed the self-assembled network formation. In conclusion, co-cultures upregulated the expression of VEGF₁₆₅ in co-cultured HBMSC; VEGF₁₆₅ then activated uPA in co-cultured HUVEC, which might be responsible for initiating the migration and the self-assembled network formation with the participation of VE-cad. All of these results indicated that only the direct contact of HBMSC and HUVEC and their respective dialogue are sufficient to stimulate secretion of soluble factors and to activate molecules that are critical for self-assembled network formation which show a great application potential for vascularization in tissue engineering.     

Risk factors for new intramammary infections during the dry period in untreated dairy cows from herds using selective dry cow therapy

This study aimed at investigating risk factors for new intramammary infections (IMI) during the dry period in untreated cows from herds using selective dry cow antibiotic therapy (DCT). A total of 980 uninfected quarters in 347 untreated cows from 28 herds using selective DCT were included in a prospective survey. A herd-level questionnaire and an individual cow-level recording sheet were implemented to collect data on putative risk factors. Quarter milk samples were taken at drying-off and on day 3 after calving to assess the occurrence of new IMI during the dry period. A multivariate model including a herd effect as random and a cow effect as repeated was run at the quarter level. Interactions between risk factors and the cow infection status at drying-off (cow infected in at least one quarter v. uninfected) were checked. Three risk factors were found significantly associated with the risk for new IMI without interaction (P < 0.05): cows infected in at least one quarter at drying-off (v. uninfected cows) (relative risks (RR) = 1.58); long preceding lactation (>355 days v. shorter length) (RR = 1.62); long dry period (>65 days v. shorter length) (RR = 1.46). One risk factor acted only in interaction with the cow infection status at drying-off: in cows uninfected at drying-off, the risk for new IMI was significantly higher in cows with short teats (RR = 1.21) when compared with cows with long or normal teats, while the reverse relationship was observed in cows infected at drying-off. Risk factors can be translated in recommendations, for instance to have dry periods not longer than 2 months. Moreover, as suggested by our results, the efficacy of selective DCT towards the prevention of new IMI would be improved if all infected cows were detected and treated. Criteria to accurately identify these infected cows should be therefore further investigated.     

RNA Mimicry by the Fap7 Adenylate Kinase in Ribosome Biogenesis

During biogenesis of the 40S and 60S ribosomal subunits, the pre-40S particles are exported to the cytoplasm prior to final cleavage of the 20S pre-rRNA to mature 18S rRNA. Amongst the factors involved in this maturation step, Fap7 is unusual, as it both interacts with ribosomal protein Rps14 and harbors adenylate kinase activity, a function not usually associated with ribonucleoprotein assembly. Human hFap7 also regulates Cajal body assembly and cell cycle progression via the p53–MDM2 pathway. This work presents the functional and structural characterization of the Fap7–Rps14 complex. We report that Fap7 association blocks the RNA binding surface of Rps14 and, conversely, Rps14 binding inhibits adenylate kinase activity of Fap7. In addition, the affinity of Fap7 for Rps14 is higher with bound ADP, whereas ATP hydrolysis dissociates the complex. These results suggest that Fap7 chaperones Rps14 assembly into pre-40S particles via RNA mimicry in an ATP-dependent manner. Incorporation of Rps14 by Fap7 leads to a structural rearrangement of the platform domain necessary for the pre-rRNA to acquire a cleavage competent conformation.     

Influences of vascularization and osteogenic cells on heterotopic bone formation within a madreporic ceramic in rats

Research in biomaterials for bone reconstruction has led to elaborate osteogenic composites that combine porous ceramics with bone marrow stromal cells. The aim of this study was to evaluate the influence of direct vascularization of such composites on osteogenesis and the ability to produce a vascularized bone substitute transplant in an ectopic muscular site. Sixty-four coralline biomaterials were implanted in 32 Fisher rats under four conditions: (1) alone (reference group M, n = 16), (2) coated with bone marrow stromal cells (group MC, n = 16), (3) combined with a vascular pedicle (group MV, n = 16), or (4) coated with bone marrow stromal cells and combined with a vascular pedicle (MCV group, n = 16). The number of vessels in the pores (vessel-pore ratio) of the implants and the proportion of pores showing bone ingrowth (bone-pore ratio) were measured at 2, 4, 6, and 8 weeks on four implants of each group. Compared with the reference group, angiogenesis was higher when the biomaterial was combined with a vascular pedicle or was coated with osteoprogenitor cells. The association of both vascular pedicle and osteoprogenitor cells increased vascularization by 60 percent (p = 0.003) and osteogenesis by 62 percent (p < 0.001). A combination of both vascular pedicle and bone marrow osteoprogenitor cells in coralline implants enhances neovascularization and osteogenesis after implantation in ectopic intramuscular sites to a greater extent than either does alone.     

Effects of five days of bed rest with intermittent centrifugation on neurovestibular function

Objectives:

We tested whether intermittent short-radius centrifugation was effective for mitigating alteration in balance and gait following bed rest.

Methods:

Ten male subjects were exposed to 5 days of 6° head-down tilt bed rest with: (a) no countermeasure; (b) daily 1-g centrifugation for a continuous 30-min period; and (c) daily 1-g centrifugation for six periods of 5 min. During and after the bed rest, subjects were asked to scale the severity of neurovestibular symptoms that followed centrifugation or 80º head-up tilt. Following the bed rest, equilibrium scores were derived from anterior-posterior sway while standing on a foam pad with the eyes open or closed while making pitch head movements, and gait was evaluated by grading subjects’ performance during various locomotion tasks.

Results:

At the beginning of bed rest, one single 30-min period of centrifugation induced more severe neurovestibular symptoms than six periods of 5-min centrifugation. After bed rest, although equilibrium scores and gait performance were not significantly altered, subjects felt less neurovestibular dysfunction with orthostatic stress when centrifugation was used.

Conclusion:

Centrifugation was effective at reducing the severity of neurovestibular symptoms after bed rest, but this decrease was not different between one or multiple daily sessions.